Methods for expansion or depletion of t-regulatory cells

ABSTRACT

The invention features methods of producing compositions enriched in Tregs and methods for treating immunological disorders using these compositions. The invention also features methods for producing compositions enriched in lymphocytes and depleted of Tregs and the use of these compositions in the treatment of proliferative disorders.

BACKGROUND OF THE INVENTION

T-regulatory cells (Tregs) are a small subset of T-lymphocytes withdiverse clinical applications in transplantation, allergy, asthma,infectious diseases, graft versus host disease (GVHD), and autoimmunity.Tregs are also involved in immunotolerance in conditions such as cancer.The use of Tregs in clinical applications has been challenging becauseof their rarity in blood and the difficulty of expanding them ex vivointo homogeneous populations. Naturally occurring Tregs constitute only1-5% of total CD4+ T cells in blood and they remain largely dormantuntil activated. Therefore, the harvesting of sufficient quantities ofTregs in order to investigate their role in basic biology and forclinical medical applications relies on the ability to expand Tregs exvivo. More than a dozen protocols have been developed worldwide toexpand Tregs ex vivo for reinfusion into patients, but all of theseprotocols produce heterogeneous progeny consisting of phenotypically andfunctionally mixed populations of CD4+ T cells. Heterogeneous CD4+ Tcell populations hold risk because they are capable of releasingpro-inflammatory cytokines and they possess cells with diverse,sometimes antagonistic functions.

Heterogeneous populations of CD4+ T cells are deemed by regulatoryagencies to be impure and irreproducible, so no clinical trials haveproceeded beyond Phase I studies. Thus, a key research and clinical goalhas been to find methods to selectively expand Tregs without stimulatingexpansion of other CD4+ T cell populations. A parallel goal in thisfield has been to find methods to selectively deplete Tregs and toexpand lymphocyte populations. Such lymphocyte populations would beuseful to upregulate the immune response in therapies for proliferativedisorders, such as cancers.

SUMMARY OF THE INVENTION

The invention features a composition enriched in CD4+CD25^(hi) Tregulatory cells (Tregs) in which at least 60% (e.g., 70%, 80%, 90%, or100%) of the cells in the composition are Tregs. Preferably, thecomposition includes a homogeneous population of Tregs with desirableimmune modulating properties, e.g., expression of forkhead box P3(FOXP3) protein. The composition also includes at least 5×10⁶ (e.g.,5×10⁷, 5×10⁸, 5×10⁹, 5×10¹⁰, 5×10¹¹, or 5×10¹²) Tregs. The Tregs in thecomposition can be characterized as positive for the expression of oneor more proteins selected from the group consisting of CTLA4, TNFR2,FOXP3, CD62L, Fas, HLA-DR, and CD45RO, and as low or negative for theexpression of one or more proteins selected from the group consisting ofCD127, CCR5, CCR6, CCR7, CXCR3, IFN-gamma, IL10, and ICOS.

The invention also features a method for producing a compositionenriched in CD4+CD25^(hi) Tregs, such as the composition describedabove. This method generally includes contacting in vitro a populationof human cells that include T lymphocytes (e.g., CD4+ cells, CD25+cells, or CD4+CD25+ cells) with a tumor necrosis factor receptor 2(TNFR2) agonist and/or an NF-κB activator (e.g., during one or moreculturing steps), thereby producing a composition that is enriched inthe CD4+CD25^(hi) Tregs. The population of human cells can be obtainedfrom a human blood sample or a human bone marrow sample from a patient.The population of human cells from the sample are, or can include, CD4+cells, CD25+ cells, or CD4+CD25+ cells, which can be isolated orenriched from the blood or bone marrow sample prior to contacting withthe TNFR2 agonist and/or the NF-κB activator. The TNFR2 agonist and/orthe NF-κB activator promote enrichment of the CD4+CD25^(hi) Tregs,according to the method, by promoting an increase in the proliferationof CD4+CD25^(hi) Tregs present in the population of human cells and/orby increasing the development of CD4+CD25^(hi) Tregs from T lymphocytes(e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells) present in thepopulation of human cells (e.g., by differentiation or activation). Themethod described above preferably produces a homogenous population ofTregs, e.g., where at least 60% (e.g., 70%, 80%, 90%, or substantially100%) of the cells in the composition are Tregs.

The TNFR2 agonist that can be used in the methods of the invention canbe an agent selected from the group consisting of an antibody (e.g., amonoclonal anti-TNFR2 antibody), a peptide, a small molecule, and aprotein. Because TNFR2 signaling can proceed via the downstream NF-κBpathway, an NF-κB activator can be used to contact the population ofhuman cells in order to produce the composition enriched in Tregs. TheNF-κB activator can be selected from the group consisting of a smallmolecule (e.g., betulinic acid, topoisomerase poison VP16, anddoxorubicin), a peptide, a protein, a virus, and a small non-coding RNA.

In addition to a TNFR2 agonist and/or a NF-κB activator, the method ofproducing a composition enriched in Tregs can include contacting thepopulation of human cells (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+cells) with one or more of interleukin-2 (IL2), rapamycin, anti-CD3(e.g., an anti-CD3 antibody), and/or anti-CD28 (e.g., an anti-CD28antibody). After in vitro proliferation, the above described methods ofthe invention can produce at least 5×10⁶ (e.g., 5×10⁶, 5×10⁷, 5×10⁸,5×10⁹, 5×10¹⁰, 5×10¹¹, or 5×10¹²) Tregs in which at least 60% (e.g.,70%, 80%, 90%, or substantially 100%) of the cells in the compositionare Tregs.

The invention also features methods for treating an immunologicaldisorder (e.g., an allergy, asthma, an autoimmune disorder, GVHD, ortransplantation graft rejection) or an infectious disease (e.g., abacterial infection, a viral infection, a fungal infection, and/or aparasitic infection) in a patient (e.g., a human patient) byadministering to the patient any one or more of a composition enrichedin Tregs, a TNFR2 agonist (e.g., a monoclonal anti-TNFR2 antibody), anda NF-κB activator. For example, the method of treatment can includeadministering the composition enriched in Tregs by itself or incombination with a NF-κB activator. The composition enriched in Tregscan be produced by any method known in the art. One method of producinga composition enriched in Tregs is by using the methods of the inventiondescribed above. The TNFR2 agonist and the NF-κB activator for use inthe method of treating an immunological disorder can be any one or moreof those described above.

Allergies that can be treated by the methods of the invention can beselected from the group consisting of food allergy, seasonal allergy,pet allergy, hives, hay fever, allergic conjunctivitis, poison ivyallergy oak allergy, mold allergy, drug allergy, dust allergy, cosmeticallergy, and chemical allergy. Autoimmune disorders that can be treatedby the methods of the invention can be selected from the groupconsisting of type I diabetes, Alopecia Areata, Ankylosing Spondylitis,Antiphospholipid Syndrome, Autoimmune Addison's Disease, AutoimmuneHemolytic Anemia, Autoimmune Hepatitis, Behcet's Disease, BullousPemphigoid, Cardiomyopathy, Celiac Sprue-Dermatitis, Chronic FatigueImmune Dysfunction Syndrome (CFIDS), Chronic Inflammatory DemyelinatingPolyneuropathy, Churg-Strauss Syndrome, Cicatricial Pemphigoid, CRESTSyndrome, Cold Agglutinin Disease, Crohn's Disease, Essential MixedCryoglobulinemia, Fibromyalgia-Fibromyositis, Graves' Disease,Guillain-Barré, Hashimoto's Thyroiditis, Hypothyroidism, IdiopathicPulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgANephropathy, Juvenile Arthritis, Lichen Planus, Lupus, Ménière'sDisease, Mixed Connective Tissue Disease, Multiple Sclerosis, MyastheniaGravis, Pemphigus Vulgaris, Pernicious Anemia, Polyarteritis Nodosa,Polychondritis, Polyglandular Syndromes, Polymyalgia Rheumatica,Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, PrimaryBiliary Cirrhosis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis, Scleroderma,Sjögren's Syndrome, Stiff-Man Syndrome, Takayasu Arteritis, TemporalArteritis/Giant Cell Arteritis, Ulcerative Colitis, Uveitis, Vasculitis,Vitiligo, and Wegener's Granulomatosis.

The above described methods of treatment can include administering acomposition enriched in Tregs that includes at least 5×10⁶ (e.g., 5×10⁶,5×10⁷, 5×10⁸, 5×10⁹, 5×10¹⁰, 5×10¹¹, or 5×10¹²) Tregs. Tregs havingdesirable immune-modulating properties include those expressing, e.g.,FOXP3.

The invention also features an isolated antibody or antigen-bindingfragment thereof that selectively binds to a first epitope of TNFR2, thefirst epitope includes positions 48-67 of SEQ ID NO: 1. The antibody orantigen-binding fragment thereof has an antagonistic effect on TNFR2upon binding. The antibody or antigen-binding fragment thereof canfurther bind to a second epitope of TNFR2. The second epitope includesposition 135 of SEQ ID NO: 1. The second epitope can include positions135-147 of SEQ ID NO: 1 (e.g., positions 130-149 of SEQ ID NO: 1,positions 128-147 of SEQ ID NO: 1, or positions 135-153 of SEQ ID NO:1). The antibody or antigen-binding fragment thereof can be a monoclonalantibody or antigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, an Fab, a humanized antibody orantigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a monovalent antibody orantigen-binding fragment thereof, a chimeric antibody or antigen-bindingfragment thereof, a single-chain Fv molecule, a bispecific single chainFv ((scFv′)₂) molecule, a domain antibody, a diabody, a triabody, anaffibody, a domain antibody, a SMIP, a nanobody, a Fv fragment, a Fabfragment, a F(ab′)₂ molecule, or a tandem scFv (taFv) fragment. Theequilibrium dissociation constant (“K_(D)”) for binding of the antibodyor antigen-binding fragment thereof to TNFR2 can be less than about 50nM (e.g., less than about 30 nM, less than about 20 nM, less than about10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM,less than about 900 pM, less than about 800 pM, or less than about 700pM). The equilibrium dissociation constant (“K_(D)”) for binding of theantibody or antigen-binding fragment thereof to TNFR2 can be in therange of about 10 pM to about 50 nM (e.g., about 20 pM to about 30 nM,about 50 pM to about 20 nM, about 100 pM to about 5 nM, about 150 pM toabout 1 nM, or about 200 pM to about 800 pM).

The invention also features a composition enriched in lymphocytes anddepleted of Tregs, in which less than 10% (e.g., less than 9%, 8%, 7%,5%, or 2% or substantially none) of the cells in the composition areTregs. This composition can be produced by any method known in the art.

Furthermore, the invention also features methods for producing acomposition enriched in lymphocytes and depleted of Tregs. This methodgenerally includes contacting in vitro a population of human cells thatinclude Tregs with a tumor necrosis factor receptor 2 (TNFR2) antagonistand/or an NF-κB inhibitor. The TNFR2 antagonist and/or the NF-κBinhibitor is used to suppress the proliferation of Tregs, therebyproducing a composition that is substantially depleted of Tregs. Thepopulation of human cells can be obtained from a human blood sample or ahuman bone marrow sample from a patient. The population of human cellscan include, e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells, whichcan be isolated or enriched from the blood or bone marrow sample priorto contacting with a TNFR2 antagonist and/or an NF-κB inhibitor. Themethod described above can be used to produce a composition enriched inlymphocytes (e.g., in which substantially 100% of the cells in thecomposition are lymphocytes) and in which less than 10% (e.g., less than9%, 8%, 7%, 5%, or 2% or substantially none) of the cells in thecomposition are Tregs.

The TNFR2 antagonist that can be used in the above method for producinga composition enriched in lymphocytes and depleted of Tregs can be anagent that is selected from the group consisting of an antibody (e.g., amonoclonal anti-TNFR2 antibody), a peptide, a small molecule, and aprotein. The NF-κB inhibitor that can be used in the above method can bean agent selected from the group consisting of a small molecule, apeptide (e.g., a cell penetrating inhibitory peptide), a protein, avirus, and a small non-coding RNA. For example, the NF-κB inhibitor canbe a small molecule selected from the group consisting of2-(1,8-naphthyridin-2-yl)-Phenol, 5-Aminosalicylic acid, BAY 11-7082,BAY 11-7085, CAPE (Caffeic Acid Phenethylester), Diethylmaleate, Ethyl3,4-Dihydroxycinnamate, Helenalin, Gliotoxin, NF-κB Activation InhibitorII JSH-23, NFκB Activation Inhibitor III, Glucocorticoid ReceptorModulator, CpdA, PPM-18, Pyrrolidinedithiocarbamic acid ammonium salt,(R)-MG-132, Rocaglamide, Sodium Salicylate, QNZ, MG-132[Z-Leu-Leu-Leu-CHO], Astaxanthin, (E)-2-Fluoro-4′-methoxystilbene,CHS-828, disulfiram, olmesartan, triptolide, withaferin, celastrol,tanshinone IIA, Ro 106-9920, cardamonin, BAY 11-7821, PSI, HU 211,ML130, PR 39, honokiol, CDI 2858522, andrographolide, anddithiocarbamates.

The TNFR2 antagonist can be a TNFR2 antagonist antibody that binds to afirst epitope of TNFR2. The first epitope includes the positions 48-67of SEQ ID NO: 1. The antibody or antigen-binding fragment thereof canbind to a second epitope of TNFR2. The second epitope includes theposition 135 of SEQ ID NO: 1 (e.g., positions 135-147 of SEQ ID NO: 1).The antibody or antigen-binding fragment thereof can be a monoclonalantibody or antigen-binding fragment thereof, a polyclonal antibody orantigen-binding fragment thereof, an Fab, a humanized antibody orantigen-binding fragment thereof, a bispecific antibody orantigen-binding fragment thereof, a monovalent antibody orantigen-binding fragment thereof, a chimeric antibody or antigen-bindingfragment thereof, a single-chain Fv molecule, a bispecific single chainFv ((scFv′)₂) molecule, a domain antibody, a diabody, a triabody, anaffibody, a domain antibody, a SM IP, a nanobody, a Fv fragment, a Fabfragment, a F(ab′)₂ molecule, or a tandem scFv (taFv) fragment. Theequilibrium dissociation constant (“K_(D)”) for binding of the antibodyor antigen-binding fragment thereof to TNFR2 can be less than about 50nM (e.g., less than about 30 nM, less than about 20 nM, less than about10 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM,less than about 900 pM, less than about 800 pM, or less than about 700pM). The equilibrium dissociation constant (“K_(D)”) for binding of theantibody or antigen-binding fragment thereof to TNFR2 can be in therange of about 10 pM to about 50 nM (e.g., about 20 pM to about 30 nM,about 50 pM to about 20 nM, about 100 pM to about 5 nM, about 150 pM toabout 1 nM, or about 200 pM to about 800 pM).

The invention features a method of treating a proliferative disorder(e.g., a cancer or a solid tumor) in a patient (e.g., a human patient)by administering to the patient any one or more of a compositionenriched in lymphocytes and depleted of Tregs, a TNFR2 antagonist (e.g.,a monoclonal anti-TNFR2 antibody), or an NF-xB inhibitor. For example,the method of treating proliferative disorders can include administeringthe composition enriched in lymphocytes (and depleted of Tregs) byitself or in combination with a NF-κB inhibitor. The compositionenriched in lymphocytes can be produced by any method known in the art.Preferably, the composition enriched in lymphocytes can be produced bythe methods of the invention as described above. The TNFR2 antagonistand the NF-κB inhibitor for use in the method of treating aproliferative disorder can be any one or more of those described above.

The invention features a method of treating an infectious disease (e.g.,a bacterial infection, a viral infection, a fungal infection, or aparasitic infection) in a patient by administering to the patient thecomposition enriched in lymphocytes and depleted of Tregs, a TNFR2antagonist (e.g., a monoclonal anti-TNFR2 antibody), or an NF-κBinhibitor. For example, the method of treating an infectious disease caninclude administering the composition enriched in lymphocytes (anddepleted of Tregs) by itself or in combination with a NF-κB inhibitor.The composition enriched in lymphocytes can be produced by any methodknown in the art. Preferably, the composition enriched in lymphocytescan be produced by the methods of the invention as described above. TheTNFR2 antagonist and the NF-κB inhibitor for use in the method oftreating a proliferative disorder can be any one or more of thosedescribed above.

The invention features a method of treating an infectious disease in apatient by administering to the patient an effective amount of theantibody or antigen-binding fragment thereof as described herein. Theinvention also features a method of treating a proliferative disease(e.g., a cancer) in a patient by administering to the patient aneffective amount of the antibody or antigen-binding fragment thereof asdescribed herein.

Cancers that can be treated according to the methods of the invention(e.g., by administering any one or more of a composition enriched inlymphocytes (and depleted of Tregs), a TNFR2 antagonist and/or an NF-κBinhibitor) can be selected from the group consisting of AcuteLymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute MyeloidLeukemia, Adrenocortical Carcinoma; AIDS-Related Lymphoma, AIDS-RelatedMalignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, BladderCancer; Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma, BrainStem Glioma, Visual Pathway and Hypothalamic Glioma, Breast Cancer,Bronchial Adenomas/Carcinoids, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Chronic Myeloproliferative Disorders, Clear CellSarcoma of Tendon Sheaths, Colon Cancer, Colorectal Cancer, CutaneousT-Cell Lymphoma, Endometrial Cancer, Epithelial Cancer, EsophagealCancer,Ewing's Family of Tumors, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Intraocular Melanoma, Retinoblastoma, Gallbladder Cancer, Gastric(Stomach) Cancer, Hairy Cell Leukemia, Head and Neck Cancer,Hepatocellular (Liver) Cancer, Hodgkin's Lymphoma, HypopharyngealCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, PituitaryCancer, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma,Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft TissueSarcoma, Squamous Neck Cancer , Testicular Cancer, Thyroid Cancer,Urethral Cancer, Uterine Sarcoma, and Vaginal Cancer. The solid tumorsthat can be treated with the methods of the invention can include solidtumors of the brain, lung, breast, lymphoid, gastrointestinal tract,genitourinary tract, pharynx, prostate, or ovary.

Definitions

The term “about” is used herein to mean a value that is ±10% of therecited value.

The term “antibody,” as used herein, includes whole antibodies orimmunoglobulins and any antigen-binding fragment or single chainsthereof. Antibodies, as used herein, can be mammalian (e.g., human ormouse), humanized, chimeric, recombinant, synthetically produced, ornaturally isolated. In most mammals, including humans, whole antibodieshave at least two heavy (H) chains and two light (L) chains connected bydisulfide bonds. Each heavy chain consists of a heavy chain variableregion (abbreviated herein as V_(H)) and a heavy chain constant region.The heavy chain constant region consists of three domains, C_(H)1,C_(H)2, and C_(H)3 and a hinge region between C_(H)1 and C_(H)2. Eachlight chain consists of a light chain variable region (abbreviatedherein as V_(L)) and a light chain constant region. The light chainconstant region consists of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system. Antibodies of the present inventioninclude all known forms of antibodies and other protein scaffolds withantibody-like properties. For example, the antibody can be a monoclonalantibody, a polyclonal antibody, human antibody, a humanized antibody, abispecific antibody, a monovalent antibody, a chimeric antibody, or aprotein scaffold with antibody-like properties, such as fibronectin orankyrin repeats. The antibody can have any of the following isotypes:IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgM, IgA (e.g., IgA1, IgA2, andIgAsec), IgD, or IgE.

The term “antigen-binding fragment,” as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to a specific antigen (e.g., CD21 receptor). The antigen-bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. The antibody fragments can be a Fab, Fab′2, scFv, SMIP,diabody, a triabody, an affibody, a nanobody, an aptamer, or a domainantibody. Examples of binding fragments encompassed of the term“antigen-binding fragment” of an antibody include, but are not limitedto: (i) a Fab fragment, a monovalent fragment consisting of the V_(L),V_(H), C_(L), and C_(H)1 domains; (ii) a F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the V_(H) and C_(H)1domains; (iv) a Fv fragment consisting of the V_(L) and V_(H) domains ofa single arm of an antibody, (v) a dAb including V_(H) and V_(L)domains; (vi) a dAb fragment (Ward et al., Nature 341 :544-546, 1989),which consists of a V_(H) domain; (vii) a dAb which consists of a V_(H)or a V_(L) domain; (viii) an isolated complementarity determining region(CDR); and (ix) a combination of two or more isolated CDRs which mayoptionally be joined by a synthetic linker. Furthermore, although thetwo domains of the Fv fragment, V_(L) and V_(H), are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the V_(L) and V_(H) regions pair to form monovalent molecules(known as single chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments are obtained usingconventional techniques known to those of skill in the art, and thefragments are screened for utility in the same manner as intactantibodies. Antigen-binding fragments can be produced by recombinant DNAtechniques or by enzymatic or chemical cleavage of intactimmunoglobulins.

The term “chimeric antibody” refers to an immunoglobulin or antibodywhose variable regions derive from a first species and whose constantregions derive from a second species. Chimeric antibodies can beconstructed, for example, by genetic engineering, from immunoglobulingene segments belonging to different species (e.g., from a mouse and ahuman).

The term “human antibody,” as used herein, is intended to includeantibodies, or fragments thereof, having variable regions in which boththe framework and CDR regions are derived from human germlineimmunoglobulin sequences as described, for example, by Kabat et al(Sequences of proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242,1991). Furthermore, if the antibody contains a constant region, theconstant region also is derived from human germline immunoglobulinsequences. The human antibodies may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences (i.e., a humanized antibodyor antibody fragment).

The term “humanized antibody” refers to any antibody or antibodyfragment that includes at least one immunoglobulin domain having avariable region that includes a variable framework region substantiallyderived from a human immunoglobulin or antibody and complementaritydetermining regions (e.g., at least one CDR) substantially derived froma non-human immunoglobulin or antibody.

The term “TNF-α mutein,” as used herein, refers to a polypeptide havingan amino acid sequence that differs from the amino acid sequence ofTNF-α by one or more amino acids, while retaining the ability toactivate or inhibit TNFR2. For example, a TNF-α mutein may have an aminoacid sequence with greater than 90% but less than 100% sequence identityrelative to the amino acid sequence of a reference polypeptide (TNF-α).

The term “substantially 100%” or “substantially homogeneous” as usedherein with respect to a Treg enriched composition of the inventionmeans at least 90%, 95%, 96%, 97%, 98%, or 99% or more (e.g., all) ofthe cells in the composition are Tregs.

The term “treating” as used herein means stabilizing or reducing anadverse symptom associated with a condition; reducing the severity of adisease symptom; slowing the rate of the progression of a disease;inhibiting or stabilizing the progression of a disease condition; orchanging a metric that is associated with the disease state in adesirable way.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a set of graphs showing that in a small double-blinded,placebo-controlled trial of human subjects, BCG treatment induces TNF-α(top left graph) and shortly thereafter Tregs appear in the treatedsubject (bottom left graph) versus placebo (right hand side graphs).

FIG. 1B is a set of graphs showing that in freshly isolated CD4+ cellsfrom fresh human blood, TNF-α alone does not induce FOXP3 in culture(left graph), but does induce it to higher levels when co-incubated withIL-2, compared to IL-2 alone (right graph). The data are from 14subjects (left panel) and 10 subjects (right panel).

FIG. 1C is a set of representative flow cytometry histograms thatconfirm greater intracellular induction of FOXP3 in CD4+CD25^(hi) Tregsafter co-incubation with TNF-α and IL-2 than with IL-2 alone. Figures inflow diagrams are %. [*P<0.05 or **P<0.01, by paired t-test].

FIG. 2A is a set of graphs showing that TNFR2 is preferentiallyexpressed on CD4+CD25^(hi) T cells.

FIG. 2B is a graph showing that one TNFR2 antibody induced FOXP3, actingas an agonist, and the other TNFR2 antibody suppressed FOXP3+expression, acting as antagonist.

FIG. 2C is a graph showing that in a signaling pathway assay, purifiedCD4+ cells, incubated with IL-2, the TNFR2 agonist and antagonisttrigger differences in relative downstream expression of mRNA,especially in signaling proteins TRAF2, TRAF3 and apoptosis inhibitorclAP2 that were preferentially induced by TNFR2 agonism. Datarepresented are means±SEM from 4 subjects.

FIG. 2D is a set of graphs showing that TNFR2 agonist triggers greater %increase in proliferation in samples from 6 subjects measured by flowcytometry (left panel) and with carboxyfluorescein diacetatesuccinimidyl ester (CFSE) measurements (right panels) and representativeresults from a typical experiment is presented with CFSE measurements(right panels). The numbers in a bar represent the percentage of cellsthat went into division. The TNFR2 antagonist suppressed CD4+proliferation (left panel) and inhibited expansion AS measured by CFSEdilution (right panel). *P<0.05 or ***P<0.01, by paired t-test.

FIG. 3A is a schematic showing the protocol for purifying CD4+CD25^(hi)cells from CD4+ cells from fresh blood and expanding for 16 days byincubation in 96 well round-bottom plate (2×10⁴ cells/well) withanti-CD3 and anti-CD28 antibodies, human IL-2, and rapamycin.

FIG. 3B is a set of graphs showing representative CD25 and FOXP3 flowdiagrams of CD4+ cells before versus after CD25^(hi) purification andexpansion, indicating purity of populations.

FIG. 3C is a graph showing cell counts of purified Tregs, by treatmentgroup, reveal that TNFR2 agonist induced more expansion than any othergroup. *p<0.05, **p<0.01, by paired t-test. The TNFR2 antagonistsuppressed expansion versus no treatment. Data in FIG. 3C are samplesfrom 10 subjects.

FIG. 4A is a set of graphs showing that all treatment groups are highlypositive for Treg markers such as CD25, FOXP3, CTLA4, TNFR2, CD62L, andFas and negative for CD127.

FIG. 4B is a set of graphs showing that TNFR2 agonist-treated Tregsalmost uniformly express HLA-DR and CD45RO and almost uniformly lackmarkers such as ICOS, CXCR3, CCR5, CCR6, CCR7, and CXCR3.

FIG. 4C is a set of representative flow diagrams showing that TNFR2agonist-treated Tregs have greater uniformity of Treg markers than doother groups (* p<0.05, **p<0.01 by t-test).

FIG. 5A is a set of graphs showing that in a representative case, TNFR2agonist-treated Tregs exerted stronger and dose-dependent suppression ofCD8+ cell numbers, compared to other groups, at all dilutions orsuppression ratios (left panel, third column). Using a suppression indexof 2:1 (CD8+ Responders to Tregs), TNFR2 agonist suppression of CD8+cells is greater than no treatment and TNFR2 antagonist treatment (rightpanel). Data in FIG. 5A (right panel) are samples from 5 subjects anddata in FIG. 5B (lower panel) are from 8 subjects.

FIG. 5B is a set of graphs showing that TNFR2 agonist-treated Tregsproduce a lower percentage of IFNγ+ cells.

FIG. 5C is a graph showing lower numbers of T-bet+ cells afterstimulation with PMA and ionomycin for 24 hours.

FIG. 6 is a schematic showing the summary of findings with TNFR2 agonistversus antagonist. After purification and expansion, the TNFR2 agonistis better than TNFR2 antagonist at proliferating and yielding morephenotypically homogeneous Tregs (CD4+ CD25^(hi) FOXP3+ CTLA4+ TNFR2+CD45RO+ CD62L+ CD127−, HLA-DR^(hi) CCR5− CCR7− CXCR3− ICOS−), withhigher suppression capacity for CD8+ cells, and lower cytokine-producingcapability.

FIG. 7 is a graph showing the induction of FOXP3 expression by differentTNFR agonists and antagonist antibodies. Screening anti-TNFR1 andanti-TNFR2 mAbs reveals that not all of the antibodies tested induce orinhibit FOXP3+ expression.

FIG. 8 is a graph showing the proportion of CD25+FOXP3− cells after IL-2overnight incubation with and without the TNFR2 agonist or antagonist.Significant percentage increases were observed if the treatment groupwas incubated in the presence of TNF or TNFR2 agonist. (**; p<0.001).Data are of samples from 10 subjects.

FIG. 9A is a schematic showing the expansion protocol. After 16 days ofexpansion, Tregs Expander Beads were removed and rested overnight forcell counting.

FIG. 9B is a graph showing the magnitude of expansion by each treatmentgroup. (*; p<0.05, by paired t-test). Data in FIG. 9B are of samplesfrom10 subjects.

FIG. 10A is a set of graphs showing that all cells were positive forFas.

FIG. 10B is a set of graphs showing that some surface markers showeddiverse patterns of expression according to the way the cells wereexpanded with a similar trend compared to cells expanded usingrapamycin. (*; p<0.05, **; p <0.01) determined by paired t test). Dataare from 6 subjects.

FIG. 11A is a graph showing the phenotypes of freshly separated Tregsbefore expansion (N=3, samples from 3 subjects).

FIG. 11B is a set of graphs showing representative flow diagrams ofTregs markers before expansion.

FIG. 12 is a set of graphs showing the density of cell surface markersmeasured by Mean Fluorescence Intensity (MFI). MFI of Tregs demonstratesclear differences between TNFR2 agonist expanded cells and TNFR2antagonist expanded cells. (*p<0.05, **p<0.01 determined by paired ttest).

FIG. 13A is a graph showing that the suppression capacity of expandedCD4+CD25+ cells was determined by CFSE dilution of CD8+ T respondercells. Flow cytometric figures of a typical result and summary ofsuppression index calculated based upon Responder:Treg of 2 : 1 fromfour independent experiments is also shown in FIG. 13A.

FIG. 13B is a set of graphs showing that CD4+CD25+ cells expanded withTNFR2 agonist exhibited significant enhanced suppression capacity (N=5).Those cells showed lowest cytokine producing capacity. (IFN, IL-10 andTNF after stimulation with PMA and ionomycin for 24 hours (*; p<0.05,**; p<0.01, by paired t test).

DETAILED DESCRIPTION OF THE INVENTION

The invention features methods for the production of a compositionenriched in Tregs (e.g., CD4+,CD25^(hi) Tregs). The invention alsofeatures methods for treating immunological disorders and infectiousdiseases using a composition enriched in Tregs, e.g., a compositionenriched in Tregs prepared by the methods described below. The inventionalso features methods for producing a composition enriched inlymphocytes (and depleted of Tregs) and methods of treatingproliferative disorders using this composition.

Tregs and TNFR2

T regulatory cells (Tregs) are a small subset of T-lymphocytes withdiverse clinical applications in transplantation, allergy, asthma,infectious diseases, GVHD, and autoimmunity. The Tregs can be used tosuppress the abnormal immune response in patients in need thereof. Tregsare also known to be involved in immunotolerance in conditions such ascancer. Naturally occurring Tregs constitute only 1-5% of total CD4+ Tcells in blood, and remain largely dormant until activated. In humans,Tregs are defined by co-expression of CD4+ and high expression of theinterleukin-2 (IL-2) receptor alpha chain CD25^(hi). Tregs also featureinducible levels of intracellular transcription factor FOXP3 and theexpression of FOXP3 can be used to identify Tregs. TNF-α has tworeceptors, TNFR1 and TNFR2, each of which controls different signalingpathways. Unlike TNFR1, which has ubiquitous cellular expression, TNFR2is expressed in a more limited manner, restricted primarily tosubpopulations of T cells (in particular, Tregs), endothelial cells, andneurons. Research in primates suggests that TNFR2-specific ligands arelikely to have minimal systemic toxicity because of the restrictedcellular distribution of TNFR2. Naturally occurring Tregs appear toexpress TNFR2 at a higher density than TNFR1. These features make TNFR2an advantageous molecular target on Tregs.

Methods for Producing an Enriched Treg Composition

The invention features methods for expanding Tregs in a sample isolatedfrom a patient (e.g., a human), such as a peripheral blood sample or abone marrow sample, to produce a composition enriched in Tregs that arecharacterized as CD4+ and CD25^(hi). Methods for promoting theproliferation of Tregs are known in the art, e.g., as described inBrunstein, C. G. et al., Infusion of ex vivo expanded T regulatory cellsin adults transplanted with umbilical cord blood: safety profile anddetection kinetics, Blood 117, 1061-1070 (2011); Saas, P. & Perruche,S., (F1000 Immunology, 2012), Tresoldi, E. et al., Stability of humanrapamycin-expanded CD4+CD25+ T regulatory cells, Haematologica 96,1357-1365 (2011); Nadig, S. N. et al., In vivo prevention of transplantarteriosclerosis by ex vivo-expanded human regulatory T cells. Nat Med16, 809-813 (2010); Battaglia, M., Stabilini, A. & Tresoldi, E.,Expanding human T regulatory cells with the mTOR-inhibitor rapamycin,Methods Mol Biol 821, 279-293 (2012); Pahwa, R. et al., Isolation andexpansion of human natural T regulatory cells for cellular therapy,Journal of immunological methods 363, 67-79 (2010); Hoffmann, P., Eder,R., Kunz-Schughart, L. A., Andreesen, R. & Edinger, M. , Large-scale invitro expansion of polyclonal human CD4(+)CD25high regulatory T cells,Blood 104, 895-903 (2004); Lin, C. H. & Hunig, T., Efficient expansionof regulatory T cells in vitro and in vivo with a CD28 superagonist,European journal of immunology 33, 626-638 (2003); Lan, Q. et al.,Induced FOXP3(+) regulatory T cells: a potential new weapon to treatautoimmune and inflammatory diseases? Journal of molecular cell biology4, 22-28 (2012); Sagoo, P. et al., Human regulatory T cells withalloantigen specificity are more potent inhibitors of alloimmune skingraft damage than polyclonal regulatory T cells, Science TranslationalMedicine 83, 1-10 (2011); Edinger, M. & Hoffmann, P., Regulatory T cellsin stem cell transplantation: strategies and first clinical experiences,Current opinion in immunology 23, 679-684 (2011); Trzonkowski, P. etal., First-in-man clinical results of the treatment of patients withgraft versus host disease with human ex vivo expanded CD4+CD25+CD127-Tregulatory cells, Clinical Immunology 133, 22-26 (2009); Di lanni, M. etal., Tregs prevent GVHD and promote immune reconstitution inHLA-haploidentical transplantation, Blood 117, 3921-3928 (2011); Hippen,K. L. et al, Massive ex vivo expansion of human natural regulatory Tcells (T(regs)) with minimal loss of in vivo functional activity. SciTransl Med 3, 83ra41 (2011); Kim, Y. C. et al., Oligodeoxynucleotidesstabilize Helios-expressing Foxp3+ human T regulatory cells during invitro expansion, Blood 119, 2810-2818 (2012); Bacchetta, R. et al.,Interleukin-10 Anergized Donor T Cell Infusion Improves ImmuneReconstitution without Severe Graft-Versus-Host-Disease AfterHaploidentical Hematopoietic Stem Cell Transplantation. ASH AnnualMeeting Abstracts 114, 45-(2009); Desreumaux, P. et al., Safety andefficacy of antigen-specific regulatory T-cell therapy for patients withrefractory Crohn's disease, Gastroenterology 143, 1207-1217 e1202(2012); Clerget-Chossat, N. et al., in International Society for CellTherapy Seattle, Wash., (2012); and Cardenas, P. A., Huang, Y. &Ildstad, S. T., The role of pDC, recipient T(reg) and donor T(reg) inHSC engraftment: Mechanisms of facilitation, Chimerism 2, 65-70 (2011).Each of these publications, and their methods for expanding Tregs, isincorporated herein by reference.

The protocols for promoting the proliferation of Tregs that aredescribed in the above publications generally include obtaining freshsample (e.g., a blood sample) from a patient (e.g., a human patient)that includes a population of CD4+ cells. The CD4+ cells can be furtherpurified or enriched in one or more steps prior to the expansion ofTregs. The CD4+ cells can be separated from the sample using techniquesknown in the art (e.g., using magnetic beads conjugated to anti-CD4+antibodies such as Dynabeads® CD4 Positive Isolation kit (Invitrogen)).During culturing, the CD4+ cells can be contacted with one or morereagents to stimulate their proliferation. For example, one or more ofanti-CD3 antibody, anti-CD28 antibody, human IL-2, and rapamycin can beadded. However, this method alone produces a heterogeneous population ofcells, some of which are capable of releasing pro-inflammatory cytokinesthat can be detrimental to the patient. This heterogeneous population isnot useful for treatment of immunological disorders or infectiousdiseases and Tregs cannot be easily isolated from this heterogeneouspopulation without damaging them. The present invention improves uponthese protocols by using a TNFR2 agonist that preferentially promotesproliferation of Tregs and produces a homogeneous population of Tregswith desirable traits, e.g., a sub-population of Tregs that expressFOXP3. The TNFR2 agonist and/or the NF-κB activator promote enrichmentof the CD4+CD25^(hi) Tregs, according to the method, by promoting anincrease in the proliferation of CD4+CD25^(hi) Tregs present in thepopulation of human cells and/or by increasing the development ofCD4+CD25^(hi) Tregs from T lymphocytes (e.g., CD4+ cells, CD25+ cells,or CD4+CD25+ cells) present in the population of human cells (e.g., bydifferentiation or activation). The invention produces a population ofcells enriched in Tregs that can be used for treatment of immunologicaldisorders or infectious diseases as described herein.

In vitro Expansion of Tregs Using a TNFR2 Agonist

In general, the present method includes obtaining a starting populationof T lymphocytes (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells)from a human sample, e.g., blood or bone marrow sample. When bloodsample is used, typically 3-4 tubes of blood (˜2-10 mL in each tube) canbe used in the protocol described below. One skilled in the art canadjust the amount of the blood sample that is used depending on thescale of the cell-culture protocol.

In general, anti-CD4 and/or anti-CD25 antibodies attached to a beadmatrix, e.g., a magnetic bead (such as Dynabeads®), can be used forisolating the CD4+ cells, CD25+ cells, or CD4+CD25+ cells. For example,the CD4+ T cells can be isolated by using commercially availablereagents, e.g., Dynabeads® CD4 Positive Isolation kit, and the CD25+cells can be isolated using commercially available reagents, e.g.,Dynabeads CD25 and/or DETACHaBEAD CD4/CD8 (Invitrogen). When CD4+CD25+cells are used as the starting population, the cells can be isolatedusing both anti-CD4 and anti-CD25 beads in a single step or in a twostep method by isolating the CD4+ cells first followed by isolation ofthe CD25+ cells, or vice versa.

The isolated CD4+ cells, CD25+ cells, or CD4+CD25+ cells can then beexpanded in cell culture for about 16 days (e.g., about 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, or 18 or more days) in a suitable cellculture vessel, e.g., a 96 well round-bottom plate (2×10⁴ cells/well),in the presence of one or more of anti-CD3 and/or anti-CD28 antibodies.The amount of cells to be used in the starting culture will depend onthe volume of the cell culture vessel. The anti-CD3 and anti-CD28antibodies can be present throughout the course of cell culture, e.g.,for each days of culture or only during a portion of the cell culture(one or several days during culturing). Typically the anti-CD3 andanti-CD28 antibodies can be added in the form of commercially availableDynabead Human Treg Expander (Invitrogen) at a bead to cell ratio of2:1.

Human IL-2 and/or rapamycin can also be added to the cell culture media,e.g., one or more times during the course of cell culture. For example,human IL-2 can be added every two days, e.g., at day 2, 4, 7, 9, 11, and14 of a 16 day cell culture period. Rapamycin can be added at day 0, 2,4, and 7 of a 16 day-cell culture period. Human IL-2 and rapamycin canbe added together or on alternating days. Typically, human IL-2 is addedtwo days after the start of cell culture. Human IL-2 and/or rapamycincan also remain in the cell culture for the entire period of theexpansion protocol. The cell culture media can be changed every 2-3 daysby changing half of the media with fresh media; the fresh media may alsocontain human IL-2 and/or rapamycin. For example, half of the media canbe changed every 2-3 days containing rapamycin (until day 7) and IL-2.Typically rapamycin can be used at a concentration of 0.5 nM to 100 μM(e.g., 0.5 nM, 1 nM, 10 nM, 50 nM, 100 nM, 200 nM, 0.5 μM, 0.75 μM , 1μM, 1.2 μM, or 2 μM). and human IL-2 can be used at a concentration of0.05 to 6,000 U/ml (e.g., 0.05 U/ml, 1 U/ml, 2 U/ml, 10 U/ml, 20 U/ml,50 U/ml, 100 U/ml, 150 U/ml, 200 U/ml, 250 U/ml, or 300 U/ml). Duringthe course of the cell culture, as described above, the cells can bepassaged as necessary. Protocols for passaging of cells are known in theart.

A TNFR2 agonist can be added at the start of culture at day 0 or atlater time points after initiation of the culture (e.g., on any one ofthe days after initiation of culturing, so long as at least one or moredays (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or16, or more days) of culturing includes contacting the cells with aTNFR2 agonist). Additional TNFR2 agonist can be added at several pointsduring the course of cell culture, e.g., at one or more of days 7, 8, 9,10, 11, or 12. Typically a TNFR2 agonist is added on day 0 andadditional TNFR2 agonist can be added on day 9 of a 16 day-cell cultureperiod (see, e.g., FIG. 3A). The TNFR2 agonist that can be typicallyused is an anti-TNFR2 monoclonal antibody. Additional TNFR2 agoniststhat can be used in this method are described below. In general, theanti-TNFR2 antibody can be used at a concentration in the range of 0.05μg/ml to 500 μg/ml, or more if necessary (e.g., 0.05 μg/ml, 0.1 μg/ml,0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 1.5 μg/ml, 2 μg/ml, 2.5 μg/ml, 3 μg/ml,3.5 μg/ml, 4 μg/ml, 4.5 μg/ml, or 5 μg/ml, 10 μg/ml, 50 μg/ml, 100μg/ml, 200 μg/ml, 300 μg/ml, 400 μg/ml, or 500 μg/ml). The anti-TNFR2antibody can be attached to a matrix, e.g., a bead, such as a magneticbead, for removal at the end of the cell culture period.

The cells can be harvested and the anti-CD3 and anti-CD28 regents can beremoved, e.g., by removal of the Treg Expander beads by theDetach-a-bead reagent or by multiple rounds of proliferation thatpermits bead detachment. Cells can then be washed in an appropriatemedium and rested. Cells can then be analyzed for expression of variousprotein markers, stored appropriately, and/or used in methods fortreating various disorders as described below.

After in vitro proliferation, the Tregs in the enriched compositioncomprise at least 60% (e.g., 70%, 80%, 90%, or 100%) of the cells in thecomposition. The method described above preferably produces a homogenouspopulation of Tregs, e.g., where substantially 100% (e.g., at least 90%,95%, 96%, 97%, 98%, or 99% or more (e.g., all)) of the cells in thecomposition are Tregs.

The method described above can result in an approximately 2 fold (e.g.,1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold or more)expansion of Tregs. The Tregs produced by this expansion protocol arecharacterized as expressing FOXP3, e.g., preferably at least 80% (e.g.,85%, 90%, 95%, 98%, 99%, or substantially 100%) of the cells in theexpanded population of Tregs express FOXP3. In addition, it ispreferable that the Tregs expanded by the methods of the inventionexpress high levels of FOXP3. The Treg population also includes a lowpercentage of cells expressing IFNγ. The Treg population also exhibitsincreased capacity for suppressing activation of CD8+ cells.

In previously described Treg expansion protocols, a disadvantage wasthat the identification of the expanded Tregs in a heterogeneouspopulation of cells required post-expansion sorting, often multiplerounds of sorting, of the Tregs. These multi-sorting procedures severelyand adversely affected the viability, function, and yield of the Tregsand therefore limited the subsequent use of the sorted cells intherapeutic applications. Furthermore, this sorting could only enrichfor Tregs expressing cell surface markers and not for Tregs expressingFOXP3, which is an intracellular marker. The present invention featuresthe proliferation of Tregs by contacting a population of human cellsthat is, or that includes, T lymphocytes (e.g., human CD4+ cells, CD25+cells, or CD4+CD25+ cells) with a TNFR2 agonist to produce asubstantially homogenous population of Tregs that express FOXP3, CD4+,and CD25^(hi). The Tregs produced by the present method require nopost-expansion sorting prior to use in therapeutic applications. This isa significant advantage over previously described Treg expansionprotocols. The Tregs produced by the present invention are also morepotent than previously described Treg cell populations, which may be aresult of their homogeneity or the subset of Tregs produced by thepresent method, or both. The present enriched Treg populations exhibithighly desirable qualities similar to those of immune-modulating Tregs.

TNFR2 Agonists

The TNFR2 agonist that can be used in the methods of the inventioninclude agents, such as an antibody, a peptide, a small molecule, and aprotein. The TNFR2 agonist is an agent that can bind to TNFR2 andactivate TNFR2 signaling. The TNFR2 agonist can be any agent that, whencontacted with CD4+ T cells, can stimulate the expression of any one ormore proteins selected from the group consisting of FOXP3, TNF, TRAF2,TRAF3, and clAP2.

In particular, the TNFR2 agonist can be a monoclonal antibody that bindsTNFR2, such as Clone MR2-1 (Cell Sciences) or Clone MAB2261 (R&DSystems, Inc.). The TNFR2 agonist can also be a TNF-α mutein that bindsonly to TNFR2 as an agonist. TNF-α muteins that can be used as TNFR2agonists include those described in, e.g., U.S. Patent ApplicationPublication No. 2008/0176796 A1; U.S. Pat. Nos. 5,486,463 and 5,422,104;PCT Publication Nos. WO 86/02381; WO 86/04606; and WO 88/06625; andEuropean Patent Nos. 155,549; 168,214; 251,037; 340,333; and 486,908.Each of these publications is incorporated herein by reference.

In addition, anti-TNFR2 antibodies that are capable of acting as TNFR2agonists are described in Galloway et al. (Eur. J. Immunol.22:3045-3048, 1992), Tartaglia et al. (J. Biol. Chem. 268:18542-18548,1993), Tartaglia et al. (J. Immunol. 151:4637-4641, 1993), Smith et al.(J. Biol. Chem. 269:9898-9905, 1994), and Amrani et al. (Am. J. Respir.Cell. Mol. Biol. 15:55-63, 1996); each of which is incorporated hereinby reference.

Peptides that are capable of acting as a TNFR2 agonist can include an 11amino acid TNF receptor agonist peptide (TNF₇₀-₈₀) described in Laichalket al. (Infection & Immunity 66:2822-2826, 1998), incorporated herein byreference.

Since activation of the NF-κB pathway is a downstream effect of TNFR2agonism, the Treg expansion method can instead, or in addition, includecontacting the T lymphocyte population (e.g., CD4+ cells, CD25+ cells,or CD4+CD25+ cells) with one or more activators of the NF-κB pathway inplace of a TNFR2 agonist. The NF-κB activator can be a small molecule, apeptide, a protein, a virus, or a small non-coding RNA. For example, theNF-κB activator any one of the small molecules described in Manuvakhovaet al., J. Neurosci. Res. 89: 58-72, 2011 (incorporated herein byreference). Alternatively, the NF-κB activator can be betulinic acid.The NF-κB activator can also be the topoisomerase poison VP16.Additionally the NF-κB activator can be doxorubicin.

Characterization of Tregs

The Tregs in the enriched composition produced by the above describedmethods are CD4+ and CD25^(hi) and can be characterized by the presenceor absence of one or more additional molecular markers. For example, theTregs produced by the methods of the invention may express one or moreproteins selected from the group consisting of FOXP3, CTLA4, TNFR2,CD62L, Fas, HLA-DR, and CD45RO and are considered to be “positive” forthese markers. Alternatively, the Tregs may not express, or may expressin low to near undetectable amounts, one or more proteins selected fromthe group consisting of CD127, CCR5, CCR6, CCR7, CXCR3, IFN-gamma,11_10, and ICOS and may be considered to be considered “negative” forthese markers. Preferably, the method produces a composition enriched inTregs, in which at least 90% of Tregs express H LA-DR and less than 5%of Tregs express ICOS.

Treatment Using an Enriched Treg Composition of the Invention

The invention features methods for treating a variety of diseases, e.g.,immunological disorders and conditions, such as allergies, asthma,autoimmune diseases, GVHD, transplantation graft rejection, andinfectious diseases by administering a composition enriched in Tregs toa patient (e.g., a human) in need thereof. The composition enriched inTregs can be produced by the methods described above, for example, bycontacting a human sample, e.g., a blood or bone marrow sample,containing T lymphocytes (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+cells) with a TNFR2 agonist (e.g., a TNFR2 agonist antibody) and/or anNF-κB activator to produce the composition enriched in Tregs (e.g., asubstantially homogenous population of Tregs). The TNFR2 agonist and/orthe NF-κB activator promote enrichment of the CD4+CD25^(hi) Tregs,according to the method, by promoting an increase in the proliferationof CD4+CD25^(hi) Tregs present in the population of human cells and/orby increasing the development of CD4+CD25^(hi) Tregs from T lymphocytes(e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells) present in thepopulation of human cells (e.g., by differentiation or activation).

A patient in need of treatment for an immunological disorder orcondition, such as an allergy, asthma, an autoimmune disease, GVHD, or atransplantation graft rejection, or for an infectious disease can beadministered an enriched composition of Tregs (e.g., CD4+CD25^(hi) Tregsor CD4+CD25^(hi)FOXP3+ Tregs) produced from their own blood or bonemarrow using the following steps: i) obtaining a cell sample, e.g., ablood or bone marrow sample, from the human patient; ii) isolating Tlymphocytes (e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells) from thesample as described in the method above; iii) subjecting these cells tothe Tregs expansion method described above (e.g., contact and/orculturing of the CD4+ cells, CD25+ cells, or CD4+CD25+ cells withanti-CD3 antibody, anti-CD28 antibody, and a TNFR2 agonist incombination with IL-2 and/or rapamycin, and/or contact or culturing ofthe CD4+ cells, CD25+ cells, or CD4+CD25+ cells with an NF-κB activator)to produce a composition enriched in Tregs (e.g., a substantiallyhomogenous population of Tregs in which the Tregs comprise, e.g., >90%of cells in the composition); and iv) introducing the compositionenriched in Tregs into the patient without any (or with limited)post-expansion sorting of the enriched Tregs in order to treat thedisease or disorder. The above steps of the method of treatment can beperformed in iterative cycles, where the number of cycles of treatmentprovided to the patient can be determined by the disorder being treated,the severity of the disorder, and/or the outcome of each treatment cycle(i.e., a change in the disease state). For example, changes in efficacymarkers and/or in clinical outcomes can be used for determining thefrequency with which blood or bone marrow should be obtained from apatient, Tregs enriched from that blood or bone marrow, and/or theenriched Tregs administered to the patient. The enriched Tregcomposition can also be stored (e.g., frozen) for future administration.

Preferably, the Tregs are obtained from a patient's own blood or bonemarrow (i.e., autologous cells), although the following methods oftreatment may also include the use of allogeneic Tregs with possiblebest fit HLA matching or from unrelated donors that have been expandedaccording to the above methods. Allogeneic Tregs preferentially share atleast 4/6 HLA markers in common with the patient receiving the enrichedTreg composition.

Treatment of an Immunological Disorder or Condition Using an EnrichedTreg Composition of the Invention

The enriched Treg composition produced by the methods described abovecan be administered to a patient suffering from an immunologicaldisorder or condition, such as an allergy, asthma, an autoimmunedisorder, GVHD, or a transplantation graft rejection, to treat theimmunological disorder or condition.

1) Allergies:

An enriched Treg composition of the invention can be used to treat oneor more allergic conditions in a patient, such as an allergy selectedfrom the group consisting of food allergy, seasonal allergy, petallergy, hives, hay fever, allergic conjunctivitis, poison ivy allergyoak allergy, mold allergy, drug allergy, dust allergy, cosmetic allergy,and chemical allergy. Administration and dosage of the Treg compositionare discussed herein below.

2) Asthma:

An enriched Treg composition of the invention can be used to treatasthma by administering the composition to a patient in need thereof.

3) Autoimmune Disorders:

An enriched Treg composition of the invention can be used to treat oneor more autoimmune disorder selected from the group consisting of type Idiabetes, Alopecia Areata, Ankylosing Spondylitis, AntiphospholipidSyndrome, Autoimmune Addison's Disease, Autoimmune Hemolytic Anemia,Autoimmune Hepatitis, Behcet's Disease, Bullous Pemphigoid,Cardiomyopathy, Celiac Sprue-Dermatitis, Chronic Fatigue ImmuneDysfunction Syndrome (CFIDS), Chronic Inflammatory DemyelinatingPolyneuropathy, Churg-Strauss Syndrome, Cicatricial Pemphigoid, CRESTSyndrome, Cold Agglutinin

Disease, Crohn's Disease, Essential Mixed Cryoglobulinemia,Fibromyalgia-Fibromyositis, Graves' Disease, Guillain-Barré, Hashimoto'sThyroiditis, Hypothyroidism, Idiopathic Pulmonary Fibrosis, IdiopathicThrombocytopenia Purpura (ITP), IgA Nephropathy, Juvenile Arthritis,Lichen Planus, Lupus, Ménière's Disease, Mixed Connective TissueDisease, Multiple Sclerosis, Myasthenia Gravis, Pemphigus Vulgaris,Pernicious Anemia, Polyarteritis Nodosa, Polychondritis, PolyglandularSyndromes, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis,Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis,Raynaud's Phenomenon, Reiter's Syndrome, Rheumatic Fever, RheumatoidArthritis, Sarcoidosis, Scleroderma, Sjögren's Syndrome, Stiff-ManSyndrome, Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis,Ulcerative Colitis, Uveitis, Vasculitis, Vitiligo, and Wegener'sGranulomatosis. Additional autoimmune diseases that can be treated withthe methods of the invention are disclosed in U.S. Pat. No. 8,173,129,incorporated herein by reference. Administration and dosage of the Tregcomposition are discussed herein below.

4) Transplantation Graft Rejection or GVHD:

An enriched Treg composition of the invention can be used to reduce orinhibit transplantation graft rejection or GVHD that occurs whentransplanted tissue is rejected by the recipients immune system. Thetransplantation graft rejection can be a chronic rejection, an acuterejection, or a hyperacute rejection. Administration and dosage of theTreg composition are discussed herein below.

In addition to the composition enriched in Tregs, other treatments thatcan be administered to the patient can include, e.g., steroid treatment,antibody-based treatment, immosuppressive drugs, blood transfer, andmarrow transplant, according to techniques known in the art.

Treatment of an Infectious Disease Using an Enriched Treg Composition ofthe Invention

The invention also features methods of treating infectious diseasescaused any one or more of a virus, a bacteria, a fungus, or a parasiteby administering a composition enriched in Tregs. The compositionenriched in Tregs can be produced by the methods described above. Themethods of the invention can be used for treating viral infectionscaused by, e.g., a member of the Flaviviridae family (e.g., a member ofthe Flavivirus, Pestivirus,and Hepacivirus genera), which includes thehepatitis C virus, Yellow fever virus; Tick-borne viruses, such as theGadgets Gully virus, Kadam virus, Kyasanur Forest disease virus, Langatvirus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus,Karshi virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjinvirus, Louping ill virus and the Negishi virus; seabird tick-borneviruses, such as the Meaban virus, Saumarez Reef virus, and the Tyuleniyvirus; mosquito-borne viruses, such as the Aroa virus, dengue virus,Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitisvirus, Murray Valley encephalitis virus, St. Louis encephalitis virus,Usutu virus, West Nile virus, Yaounde virus, Kokobera virus, Bagazavirus, Ilheus virus, Israel turkey meningoencephalo-myelitis virus,Ntaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus, EdgeHill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus,Wesselsbron virus, yellow fever virus; and viruses with no knownarthropod vector, such as the Entebbe bat virus, Yokose virus, Apoivirus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus,San Perlita virus, Bukalasa bat virus, Carey Island virus, Dakar batvirus, Montana myotis leukoencephalitis virus, Phnom Penh bat virus, RioBravo virus, Tamana bat virus, and the Cell fusing agent virus; a memberof the Arenaviridae family, which includes the Ippy virus, Lassa virus(e.g., the Josiah, LP, or GA391 strain), lymphocytic choriomeningitisvirus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal virus,Guanarito virus, Junin virus, Latino virus, Machupo virus, Oliverosvirus, Parana virus, Pichinde virus, Pirital virus, Sabia virus,Tacaribe virus, Tamiami virus, Whitewater Arroyo virus, Chapare virus,and Lujo virus; a member of the Bunyaviridae family (e.g., a member ofthe Hantavirus, Nairovirus, Orthobunyavirus, and Phlebovirus genera),which includes the Hantaan virus, Sin Nombre virus, Dugbe virus,Bunyamwera virus, Rift Valley fever virus, La Crosse virus, Californiaencephalitis virus, and Crimean-Congo hemorrhagic fever (CCHF) virus; amember of the Filoviridae family, which includes the Ebola virus (e.g.,the Zaire, Sudan, Ivory Coast, Reston, and Uganda strains) and theMarburg virus (e.g., the Angola, Ci67, Musoke, Popp, Ravn and LakeVictoria strains); a member of the Togaviridae family (e.g., a member ofthe Alphavirus genus), which includes the Venezuelan equine encephalitisvirus (VEE), Eastern equine encephalitis virus (EEE), Western equineencephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forestvirus, Ross River virus, Barmah Forest virus, O'nyong'nyong virus, andthe chikungunya virus; a member of the Poxviridae family (e.g., a memberof the Orthopoxvirus genus), which includes the smallpox virus,monkeypox virus, and vaccinia virus; a member of the Herpesviridaefamily, which includes the herpes simplex virus (HSV; types 1, 2, and6), human herpes virus (e.g., types 7 and 8), cytomegalovirus (CMV),Epstein-Barr virus (EBV), Varicella-Zoster virus, and Kaposi's sarcomaassociated-herpesvirus (KSHV); a member of the Orthomyxoviridae family,which includes the influenza virus (A, B, and C), such as the H5N1 avianinfluenza virus or H1N1 swine flu; a member of the Coronaviridae family,which includes the severe acute respiratory syndrome (SARS) virus; amember of the Rhabdoviridae family, which includes the rabies virus andvesicular stomatitis virus (VSV); a member of the Paramyxoviridaefamily, which includes the human respiratory syncytial virus (RSV),Newcastle disease virus, hendravirus, nipahvirus, measles virus,rinderpest virus, canine distemper virus, Sendai virus, humanparainfluenza virus (e.g., 1, 2, 3, and 4), rhinovirus, and mumps virus;a member of the Picornaviridae family, which includes the poliovirus,human enterovirus (A, B, C, and D), hepatitis A virus, and thecoxsackievirus; a member of the Hepadnaviridae family, which includesthe hepatitis B virus; a member of the Papillamoviridae family, whichincludes the human papilloma virus; a member of the Parvoviridae family,which includes the adeno-associated virus; a member of the Astroviridaefamily, which includes the astrovirus; a member of the Polyomaviridaefamily, which includes the JC virus, BK virus, and SV40 virus; a memberof the Calciviridae family, which includes the Norwalk virus; a memberof the Reoviridae family, which includes the rotavirus; and a member ofthe Retroviridae family, which includes the human immunodeficiency virus(HIV; e.g., types 1 and 2), and human T-lymphotropic virus Types I andII (HTLV-1 and HTLV-2, respectively)).

The methods of the invention can also be used for treating bacterialinfections. Examples of bacterial infections that may be treatedinclude, but are not limited to, those caused by bacteria within thegenera Salmonella, Streptococcus, Bacillus, Listeria, Corynebacterium,Nocardia, Neisseria, Actinobacter, Moraxella, Enterobacteriacece,Pseudomonas, Escherichia, Klebsiella, Serratia, Enterobacter, Proteus,Salmonella, Shigella, Yersinia, Haemophilus, Bordatella, Legionella,Pasturella, Francisella, Brucella, Bartonella, Clostridium, Vibrio,Campylobacter, and Staphylococcus.

The methods of the invention can also be used for treating parasiticinfections caused by a protozoan parasite (e.g., an intestinal protozoa,a tissue protozoa, or a blood protozoa) or a helminthic parasite (e.g.,a nematode, a helminth, an adenophorea, a secementea, a trematode, afluke (blood flukes, liver flukes, intestinal flukes, and lung flukes),or a cestode). Exemplary protozoan parasites include Entamoebahystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatidagambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi,Leishmania mexicana, Leishmania braziliensis, Leishmania tropica,Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodiumovale, Plasmodium malariae, Plasmodium falciparum, Trichomonasvaginalis, and Histomonas meleagridis. Exemplary helminthic parasitesinclude richuris trichiura, Ascaris lumbricoides, Enterobiusvermicularis, Ancylostoma duodenale, Necator americanus, Strongyloidesstercoralis, Wuchereria bancrofti, and Dracunculus medinensis,Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum,Fasciola hepatica, Fasciola gigantica, Heterophyes heterophyes, andParagonimus westermani, Taenia solium, Taenia saginata, Hymenolepisnana, and Echinococcus granulosus.

The methods of the invention can also be used for treating fungalinfections. Examples of fungal infections that may be treated include,but are not limited to, those caused by, e.g., Aspergillus, Candida,Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor,Pneumocystis, and Absidia.

Administration and dosage of the Treg composition in methods fortreating an infectious disease are discussed herein below.

Treatment Using an NF-κB Activator

Because TNFR2 signaling is transduced via activation of the NF-κBpathway, activators of NF-κB signaling can also be used in place of, orin combination with, administration of an enriched Treg composition fortreating immunological disorders or conditions and infectious diseasesaccording to the methods described above. For example, any one of theNF-κB activators described above can be used in the methods of treatmentdescribed above. The NF-κB activator can be used by itself or incombination with the composition enriched in Tregs.

Methods for Producing an Enriched Lymphocyte Composition Depleted ofTregs

The invention also features methods for producing a composition enrichedin lymphocytes (and depleted of Tregs) in vitro. Preferably the methodproduces a composition in which less than 10% of the cells (e.g., lessthan 10%, 5%, 3%, 1%, or 0.5%, or none of the cells) in the compositionare Tregs. The method is similar to the method for producing acomposition enriched in Tregs except a TNFR2 antagonist, such as ananti-TNFR2 monoclonal antibody, is used in place of a TNFR2 agonist.Additional TNFR2 antagonists that can be used in this method aredescribed below.

The method generally involves the separation of T lymphocytes (e.g.,CD4+ cells, CD25+ cells, or CD4+CD25+ cells) from human samples, e.g.,human blood or bone marrow sample, followed by expansion of the cellsduring culturing by incubation of the cells with anti-CD3 and anti-CD28antibodies. During the expansion step, the cells are contacted with aTNFR2 antagonist. The TNFR2 antagonist suppresses the proliferation ofTregs in the culture, thereby producing a composition that is enrichedin lymphocytes and depleted of Tregs. Human-IL-2 and/or rapamycin mayoptionally be added to the cell culture during the expansion of cells.

After in vitro enrichment of lymphocytes (and depletion of Tregs), lessthan 10% (e.g., less than 10%, 9%, 8%, 7%, 5%, or 2% or substantiallynone) of the cells in this composition are Tregs. The method describedabove can result in approximately 2 fold (e.g., 2.5-fold, 3-fold,3.5-fold, or 4-fold or more) enrichment of non-Treg lymphocytes (e.g.,CD4+ T cells, CD8+ T cells, CD4+ CD8+ T cells, B cells, natural killercells, etc.). The enriched lymphocyte population may also includedendritic cells, monocytes, macrophages, and neutrophils.

TNFR2 Antagonists

The TNFR2 antagonist that can be used in this method of the inventioncan include agents, such as an antibody, a peptide, a small molecule,and a protein that can bind to TNFR2 and suppress TNFR2 signaling. TheTNFR2 antagonist can be an agent that, when contacted with CD4+ T cells,can stimulate the expression of clAP but not the expression of TRAF2,TRAF3, or FOXP3.

The TNFR2 antagonist can be a monoclonal antibody that binds TNFR2.There are two epitopes of TNFR2 that the TNFR2 antagonist antibody canbind. The first epitope includes positions 48-67 (QTAQMCCSKCSPGQHAKVFC)of SEQ ID NO: 1 (amino acid sequence of human TNFR2). The second epitopeincludes position 135 (R) of SEQ ID NO: 1 (e.g., positions 135-153(RLCAPLRKCRPGF) of SEQ ID NO: 1). For example, the TNFR2 antagonistantibody can be any one of Clone MAB726 (R&D Systems, Inc.) or Clone M1(BD Biosciences). While each MAB726 and M1 binds the second epitope, anantibody of the invention may bind the first epitope or both epitopes.The TNFR2 antagonist antibody or antigen-binding fragment thereof canbind TNFR2 with a K_(D) of less than about 50 nM (e.g., less than about30 nM, less than about 20 nM, less than about 10 nM, less than about 5nM, less than about 2 nM, less than about 1 nM, less than about 900 pM,less than about 800 pM, or less than about 700 pM). The TNFR2 antagonistantibody or antigen-binding fragment thereof can bind TNFR2 with a K_(D)in the range of about 10 pM to about 50 nM (e.g., about 20 pM to about30 nM, about 50 pM to about 20 nM, about 100 pM to about 5 nM, about 150pM to about 1 nM, or about 200 pM to about 800 pM). The TNFR2 antagonistantibody avidity can be determined using methods known in the art (e.g.,surface plasmon resonance. For example, MAB 726 binds TNFR2 with a K_(D)of 621 pM (determined by surface plasmon resonance (Pioneer SensiQ®,Oklahoma City, Okla.)). The TNFR2 antagonist can also be a TNF-α muteinthat is capable of binding to TNFR2 and suppressing downstreamsignaling.

The TNFR2 antagonist can function via downstream signaling by inhibitionof the NF-κB pathway. Thus, the method of the invention can also includecontacting the human sample e.g., a blood or bone marrow sample, withone or more inhibitors of the NF-κB pathway in order to achieve the sameeffect as that of using a TNFR2 antagonist. The NF-κB inhibitor can be asmall molecule, a peptide, a protein, a virus, or a small non-codingRNA. In one embodiment, the NF-κB inhibitor that can be used in themethods to produce a composition enriched in lymphocytes can be any oneor more of 2-(1,8-naphthyridin-2-yl)-Phenol, 5-Aminosalicylic acid, BAY11-7082, BAY 11-7085, CAPE (Caffeic Acid Phenethylester),Diethylmaleate, Ethyl 3,4-Dihydroxycinnamate, Helenalin, Gliotoxin,NF-κB Activation Inhibitor II JSH-23, NFκB Activation Inhibitor III,Glucocorticoid Receptor Modulator, CpdA, PPM-18,Pyrrolidinedithiocarbamic acid ammonium salt, (R)-MG-132, Rocaglamide,Sodium Salicylate, QNZ, MG-132 [Z-Leu-Leu-Leu-CHO], Astaxanthin,(E)-2-Fluoro-4′-methoxystilbene, CHS-828, disulfiram, olmesartan,triptolide, withaferin, celastrol, tanshinone IIA, Ro 106-9920,cardamonin, BAY 11-7821, PSI, HU 211, ML130, PR 39, honokiol, CDI2858522, andrographolide, and dithiocarbamates. The NF-κB inhibitor canalso be a peptide inhibitor, e.g., a cell penetrating inhibitory peptideas described in May et al., Science, 2000 Sep. 1 ; 289(5484):1550-4 andin Orange and May, Cell Mol. Life Sci., 2008 November; 65(22):3564-91,each of which is incorporated herein by reference. Additional NF-κBinhibitors are also described in Gilmore and Herscovitch, Oncogene(2006) 25, 6887-6899; Nam, Mini Rev. Med. Chem., 2006 August;6(8):945-51; and in U.S. Pat. No. 6,410,516, each of which isincorporated herein by reference.

Methods of Treatment Using a Treg-Depleted, Lymphocyte-EnrichedComposition and/or Antagonist of the TNFR2 Signaling Pathway

The invention features methods for treating proliferative disorders,e.g., cancers, by administering a composition enriched in lymphocytesand depleted of Tregs to a patient in need thereof. The compositionenriched in lymphocytes can be produced by the methods described above,for example, by contacting cells obtained from a human sample, e.g.,blood or bone marrow sample, with a TNFR2 antagonist, e.g., a TNFR2antagonist antibody, and/or an NF-κB inhibitor to produce a compositionenriched in lymphocytes and depleted of Tregs. An NF-κB inhibitor may beused in methods of treating proliferative disorders, e.g., cancers,instead of the composition enriched in lymphocytes and depleted of Tregsor in combination with this composition. The invention also featuresmethods for treating proliferative disorders, e.g., cancers, byadministering a composition containing a TNFR2 antagonist (e.g., ananti-TNFR2 antagonist antibody) to a patient in need thereof.

The invention features methods for treating infectious diseases, byadministering a composition enriched in lymphocytes and depleted ofTregs to a patient in need thereof. The composition enriched inlymphocytes can be produced by the methods described above, for example,by contacting cells obtained from a human sample, e.g., blood or bonemarrow sample, with a TNFR2 antagonist, e.g., a TNFR2 antagonistantibody, and/or an NF-κB inhibitor to produce a composition enriched inlymphocytes and depleted of Tregs. An NF-κB inhibitor may be used inmethods of treating infectious diseases, instead of the compositionenriched in lymphocytes and depleted of Tregs or in combination withthis composition. The invention also features methods for treatinginfectious diseases, by administering a composition containing a TNFR2antagonist (e.g., an anti-TNFR2 antagonist antibody) alone to a patientin need thereof.

Treatment of Proliferative Disorders Using an Enriched LymphocyteComposition of the Invention

Non-Treg lymphocytes in a patient's blood can be expanded andadministered back to the patient in order to treat a proliferativedisorder (e.g., a cancer). The enriched lymphocyte composition can beadministered alone or in combination with one or more anti-cancer agentsknown in the art. The enriched lymphocyte composition can be prepared asfollows: i) obtaining a sample e.g., a blood or bone marrow sample, froma human patient and isolating nucleated cells (e.g., lymphocytes, suchas T lymphocytes, e.g., CD4+ cells, CD25+ cells, or CD4+CD25+ cells)present therein; ii) subjecting these cells to the lymphocyte expansionmethod described above to produce a composition enriched in lymphocytesand depleted of Tregs (e.g., a substantially homogenous population oflymphocytes in which Tregs comprise less than 10% of cells in thecomposition, preferably less than 5% of the cells in the composition orare absent in the expanded composition); and iii) introducing thecomposition enriched in lymphocytes into the patient without anypost-expansion sorting of the lymphocytes. The above steps of the methodof treatment can be performed in iterative cycles, where the number ofcycles of treatment provided to a patient can be determined by theproliferative disorder being treated, the severity of the disorder,and/or the outcome of each treatment cycle, i.e., a change in thedisease state. For example, changes in efficacy markers and/or inclinical outcomes can be used for determining the frequency with whichblood or bone marrow should be drawn from a patient, the lymphocytesenriched (and Tregs depleted) from that blood, and the enrichedlymphocytes administered to the patient. The enriched lymphocytecomposition may also be prepared and stored for later use (e.g.,frozen).

Preferably, the lymphocytes are obtained from a patient's own blood orbone marrow (i.e., autologous cells), although the following methods oftreatment may also include the use of allogeneic lymphocytes withpossible best fit HLA matching or from unrelated donors that have beenexpanded according to the above methods. Allogeneic lymphocytespreferentially share at least 4/6 HLA markers in common with the patientreceiving the enriched lymphocyte composition.

Treatments for proliferative disorders according to the presentinvention may include inhibiting the NF-κB signaling pathway incombination with the administration of an enriched lymphocytecomposition depleted of Tregs. Because TNFR2 signaling is transduced viathe NF-κB pathway, the treatment of proliferative disorders according tothe present invention can also include administering to a patient aninhibitor of the NF-κB pathway. For example, any one of the NF-κBinhibitors described above can be administered for treating aproliferative disorder. The NF-κB inhibitor can be administered byitself or in combination with the composition enriched in lymphocytes.The NF-κB inhibitor can also function independently of the TNFR2signaling pathway.

Proliferative disorders that can be treated by administering theenriched lymphocyte/Treg depleted composition include one or morecancers selected from the group consisting of Acute

Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute MyeloidLeukemia, Adrenocortical Carcinoma; AIDS-Related Lymphoma, AIDS-RelatedMalignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, BladderCancer; Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma, BrainStem Glioma, Visual Pathway and Hypothalamic Glioma, Breast Cancer,Bronchial Adenomas/Carcinoids, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Chronic Myeloproliferative Disorders, Clear CellSarcoma of Tendon Sheaths, Colon Cancer, Colorectal Cancer, CutaneousT-Cell Lymphoma, Endometrial Cancer, Epithelial Cancer, EsophagealCancer,Ewing's Family of Tumors, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Intraocular Melanoma, Retinoblastoma, Gallbladder Cancer, Gastric(Stomach) Cancer, Hairy Cell Leukemia, Head and Neck Cancer,Hepatocellular (Liver) Cancer, Hodgkin's Lymphoma, HypopharyngealCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, PituitaryCancer, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma,Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft TissueSarcoma, Squamous Neck Cancer, Testicular Cancer, Thyroid Cancer,Urethral Cancer, Uterine Sarcoma, and Vaginal Cancer. The proliferativedisorders can also include solid tumors including malignancies (e.g.,sarcomas, adenocarcinomas, and carcinomas) of the various organ systems,such as those of brain, lung, breast, lymphoid, gastrointestinal (e.g.,colon), and genitourinary (e.g., renal, urothelial, or testiculartumors) tracts, pharynx, prostate, and ovary. Exemplary adenocarcinomasinclude colorectal cancers, renal-cell carcinoma, liver cancer,non-small cell carcinoma of the lung, and cancer of the small intestine.

Administration and dosage of the enriched lymphocyte composition in themethod for treating a proliferative disease are discussed herein below.

Treatment of an Infectious Disease Using an Enriched LymphocyteComposition of the Invention

The invention also features methods of treating infectious diseasescaused by any one or more of a virus, bacteria, a fungus, or a parasite.The methods involve administering a composition enriched in lymphocytes(e.g., CD8+ T cells, B cells, or natural killer cells) and depleted ofTregs. This composition can be produced by the methods described above.The methods of the invention can be used for treating viral infectionscaused by, e.g., a member of the Flaviviridae family (e.g., a member ofthe Flavivirus, Pestivirus,and Hepacivirus genera), which includes thehepatitis C virus, Yellow fever virus; Tick-borne viruses, such as theGadgets Gully virus, Kadam virus, Kyasanur Forest disease virus, Langatvirus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus,Karshi virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjinvirus, Louping ill virus and the Negishi virus; seabird tick-borneviruses, such as the Meaban virus, Saumarez Reef virus, and the Tyuleniyvirus; mosquito-borne viruses, such as the Aroa virus, dengue virus,Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitisvirus, Murray Valley encephalitis virus, St. Louis encephalitis virus,Usutu virus, West Nile virus, Yaounde virus, Kokobera virus, Bagazavirus, Ilheus virus, Israel turkey meningoencephalo-myelitis virus,Ntaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus, EdgeHill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus,Wesselsbron virus, yellow fever virus; and viruses with no knownarthropod vector, such as the Entebbe bat virus, Yokose virus, Apoivirus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus,San Perlita virus, Bukalasa bat virus, Carey Island virus, Dakar batvirus, Montana myotis leukoencephalitis virus, Phnom Penh bat virus, RioBravo virus, Tamana bat virus, and the Cell fusing agent virus; a memberof the Arenaviridae family, which includes the Ippy virus, Lassa virus(e.g., the Josiah, LP, or GA391 strain), lymphocytic choriomeningitisvirus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal virus,Guanarito virus, Junin virus, Latino virus, Machupo virus, Oliverosvirus, Parana virus, Pichinde virus, Pirital virus, Sabiá virus,Tacaribe virus, Tamiami virus, Whitewater Arroyo virus, Chapare virus,and Lujo virus; a member of the Bunyaviridae family (e.g., a member ofthe Hantavirus, Nairovirus, Orthobunyavirus, and Phlebovirus genera),which includes the Hantaan virus, Sin Nombre virus, Dugbe virus,Bunyamwera virus, Rift Valley fever virus, La Crosse virus, Californiaencephalitis virus, and Crimean-Congo hemorrhagic fever (CCHF) virus; amember of the Filoviridae family, which includes the Ebola virus (e.g.,the Zaire, Sudan, Ivory Coast, Reston, and Uganda strains) and theMarburg virus (e.g., the Angola, Ci67, Musoke, Popp, Ravn and LakeVictoria strains); a member of the Togaviridae family (e.g., a member ofthe Alphavirus genus), which includes the Venezuelan equine encephalitisvirus (VEE), Eastern equine encephalitis virus (EEE), Western equineencephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forestvirus, Ross River virus, Barmah Forest virus, O'nyong'nyong virus, andthe chikungunya virus; a member of the Poxviridae family (e.g., a memberof the Orthopoxvirus genus), which includes the smallpox virus,monkeypox virus, and vaccinia virus; a member of the Herpesviridaefamily, which includes the herpes simplex virus (HSV; types 1, 2, and6), human herpes virus (e.g., types 7 and 8), cytomegalovirus (CMV),Epstein-Barr virus (EBV), Varicella-Zoster virus, and Kaposi's sarcomaassociated-herpesvirus (KSHV); a member of the Orthomyxoviridae family,which includes the influenza virus (A, B, and C), such as the H5N1 avianinfluenza virus or H1 N1 swine flu; a member of the Coronaviridaefamily, which includes the severe acute respiratory syndrome (SARS)virus; a member of the Rhabdoviridae family, which includes the rabiesvirus and vesicular stomatitis virus (VSV); a member of theParamyxoviridae family, which includes the human respiratory syncytialvirus (RSV), Newcastle disease virus, hendravirus, nipahvirus, measlesvirus, rinderpest virus, canine distemper virus, Sendai virus, humanparainfluenza virus (e.g., 1, 2, 3, and 4), rhinovirus, and mumps virus;a member of the Picornaviridae family, which includes the poliovirus,human enterovirus (A, B, C, and D), hepatitis A virus, and thecoxsackievirus; a member of the Hepadnaviridae family, which includesthe hepatitis B virus; a member of the Papillamoviridae family, whichincludes the human papilloma virus; a member of the Parvoviridae family,which includes the adeno-associated virus; a member of the Astroviridaefamily, which includes the astrovirus; a member of the Polyomaviridaefamily, which includes the JC virus, BK virus, and SV40 virus; a memberof the Calciviridae family, which includes the Norwalk virus; a memberof the Reoviridae family, which includes the rotavirus; and a member ofthe Retroviridae family, which includes the human immunodeficiency virus(HIV; e.g., types 1 and 2), and human T-lymphotropic virus Types I andII (HTLV-1 and HTLV-2, respectively)).

The methods of the invention can also be used for treating bacterialinfections. Examples of bacterial infections that may be treatedinclude, but are not limited to, those caused by bacteria within thegenera Salmonella, Streptococcus, Bacillus, Listeria, Corynebacterium,Nocardia, Neisseria, Actinobacter, Moraxella, Enterobacteriacece,Pseudomonas, Escherichia, Klebsiella, Serratia, Enterobacter, Proteus,Salmonella, Shigella, Yersinia, Haemophilus, Bordatella, Legionella,Pasturella, Francisella, Brucella, Bartonella, Clostridium, Vibrio,Campylobacter, and Staphylococcus.

The methods of the invention can also be used for treating parasiticinfections caused by a protozoan parasite (e.g., an intestinal protozoa,a tissue protozoa, or a blood protozoa) or a helminthic parasite (e.g.,a nematode, a helminth, an adenophorea, a secementea, a trematode, afluke (blood flukes, liver flukes, intestinal flukes, and lung flukes),or a cestode). Exemplary protozoan parasites include Entamoebahystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatidagambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi,Leishmania mexicana, Leishmania braziliensis, Leishmania tropica,Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodiumovale, Plasmodium malariae, Plasmodium falciparum, Trichomonasvaginalis, and Histomonas meleagridis. Exemplary helminthic parasitesinclude richuris trichiura, Ascaris lumbricoides, Enterobiusvermicularis, Ancylostoma duodenale, Necator americanus, Strongyloidesstercoralis, Wuchereria bancrofti, and Dracunculus medinensis,Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum,Fasciola hepatica, Fasciola gigantica, Heterophyes heterophyes, andParagonimus westermani, Taenia solium, Taenia saginata, Hymenolepisnana, and Echinococcus granulosus.

The methods of the invention can also be used for treating fungalinfections. Examples of fungal infections that may be treated include,but are not limited to, those caused by, e.g., Aspergillus, Candida,Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor,Pneumocystis, and Absidia.

Administration and dosage of the lymphocyte-enriched composition inmethods for treating an infectious disease are discussed herein below.

Dosage

The composition enriched in Tregs can be administered to a patient inneed thereof one or more times per day, week, month (e.g., one or moretimes every 2 weeks), or year depending on the severity of the diseaseand change in disease state of the patient during the treatment.Generally it is expected that a typical dosage would include 5×10⁵ to5×10¹² (e.g., 5×10⁵, 5×10⁶, 5×10⁷, 5×10⁸, 5×10⁹, 5×10¹⁰, 5×10¹¹, or5×10¹²) Tregs. Disease metrics, such as severity of symptoms, change insymptoms, patient response to treatment, any adverse effects oftreatment, and/or effect of any additional treatment(s), can be used todetermine the frequency of treatment and the dosage, i.e., the number ofTregs to be administered to a patient.

The composition enriched in lymphocytes (and depleted of Tregs) can beadministered one or more times per day, week, month (e.g., one or moretimes every 2 weeks), or year depending on the severity of the diseaseand change in disease state of the patient during the treatment.Preferably, less than 10% of the cells (e.g., less than 9%, 8%, 7%, 5%,%, 1% or none of the cells) in this composition enriched in lymphocytesare Tregs. Generally it is expected that a typical dosage would include5×10⁵ to 5×10¹²(e.g., 5×10⁵, 5×10⁶, 5×10⁷, 5×10⁸, 5×10⁹, 5×10¹⁰, 5×10¹¹,or 5×10¹²) cells in the enriched lymphocyte composition. Diseasemetrics, such as severity of symptoms, change in symptoms, patientresponse to treatment, any adverse effects of treatment, and/or effectof any additional treatment(s), can be used to determine the frequencyof treatment and the dosage, i.e., the number of lymphocytes to beadministered to a patient.

Administration

Generally, the compositions of the invention (e.g., the compositionenriched in Tregs or the composition enriched in lymphocytes anddepleted of Tregs) can be administered in any medically useful form. Forexample, such compositions may include the addition of compounds, e.g.,adjuvants, preservatives, carriers, excipients, diluents, anti-bacterialor anti-mycotic agents, anti-inflammatory agents, and/or anti-canceragents, where appropriate. The compositions of the invention can beadministered intravenously, intramuscularly, orally, by inhalation,parenterally, intraperitoneally, intraarterially, transdermally,sublingually, nasally, transbuccally, liposomally, adiposally,ophthalmically, intraocularly, subcutaneously, intrathecally, orally, orlocally, and they are formulated, as appropriate, depending on thechosen route of administration.

Administration of Antibodies of the Invention

Pharmaceutical compositions containing an anti-TNFR2 antibody of theinvention (e.g., an anti-TNFR2 antagonist antibody) are prepared forstorage by mixing the antibody having the desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizersin the form of aqueous solutions, lyophilized or other driedformulations. The acceptable excipient or carrier is selected on thebasis of the mode and route of administration. Suitable pharmaceuticalcarriers, as well as pharmaceutical necessities for use inpharmaceutical formulations, are described in Remington: The Science andPractice of Pharmacy, 21^(st) edition, Ed. Gennaro; Lippincott, Williams& Wilkins (2005), a well-known reference text in this field, and in theUSP/NF (United States Pharmacopeia and the National Formulary).Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, histidine and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Other exemplary excipients are described in Handbook of PharmaceuticalExcipients, 6^(th) Edition, Rowe et al., Eds., Pharmaceutical Press(2009).

The compositions of the invention may be prepared in a pharmaceuticallyacceptable carrier or excipient. Such suitable carriers or excipientsmay be selected from, for example, water, saline (e.g.,phosphate-buffered saline (PBS) or acetate-buffered saline (ABS), orRinger's solution), dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, a composition foradministration to a mammal can contain minor amounts of auxiliarysubstances, such as wetting or emulsifying agents, or pH bufferingagents that enhance the effectiveness of the composition. Thecompositions of the invention may also be prepared in any acceptablesalt formulation. Other agents that may be used in preparation of thecompositions of the invention include, e.g., adjuvants, preservatives,diluents, anti-bacterial or anti-mycotic agents, anti-inflammatoryagents, and/or anti-cancer agents, where appropriate.

The compositions may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington: The Science and Practice of Pharmacy, 21^(st) edition, Ed.Gennaro; Lippincott, Williams & Wilkins (2005).

The compositions to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin of the invention,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods.

The compositions containing one or more anti-TNFR2 antibodies (e.g.,anti-TNFR2 antagonist antibody) may be administered to a patient priorto the development of symptoms of a proliferative disease or aninfectious disease or the compositions may be administered to thepatient after diagnosis with a proliferative disease or an infectiousdisease after presentation with one or more (e.g., 1, 2, 3, 4, or 5)symptoms of the disease. The dosage of the anti-TNFR2 antibodies dependson the patient's state of health, but generally ranges from about 0.1 mgto about 400 mg of an antibody per dose (e.g., 1 mg, 5 mg, 10 mg, 20 mg,50 mg, 100 mg, 200 mg, 300 mg, or 400 mg or more per dose).

The compositions may be administered to a patient in one or more doses(e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more doses). If more than onedose is administered, the doses may be administered via the same mode ofadministration (e.g., intravenous administration) or by different modesof administration (e.g., intravenous and intramuscular administration).The patient may also be administered different doses at different times.For example, the patient may be administered a higher initial dose andlower subsequent doses over the course of treatment or vice versa.

The compositions may be administered daily, weekly, monthly, or yearly.For example, a dose of the composition may be administered twice daily,biweekly, bi-annually, tri-annually, or quarterly. The dose of thecomposition may be determined by a skilled physician upon considerationof a subject's clinical symptoms and/or physical condition (e.g.,weight, sex, height, and severity of the proliferative or infectiousdisease). The composition may be administered by intravenous,intradermal, parenteral, intra-arterial, subcutaneous, intramuscular,intraorbital, topical, intraventricular, intraspinal, intraperitoneal,intranasal, intracranial, or oral administration.

Kits of the Invention

The invention features a kit for the production of a compositionenriched in Tregs. The kit can include a TNFR2 agonist (e.g., a TNFR2agonist antibody) or an NF-κB activator (e.g., one or more of the NF-κBactivators described above), reagents and/or devices for isolating ahuman sample, e.g., a blood or bone marrow sample, reagents and/ordevices for isolating blood or bone marrow cells (e.g., CD4+CD25+ cells)from the sample, and reagents for culturing the blood or bone marrowcells (e.g., anti-CD3 antibody, anti-CD28 antibody, interleukin-2,and/or rapamycin). Additionally the kit of the invention can alsoinclude instructions for performing the method of the invention, e.g.,instructions for isolating a blood or bone marrow sample and blood orbone marrow cells therefrom, instructions for contacting blood or bonemarrow cells with a TNFR2 agonist, and/or instructions for culturing,harvesting and/or storing the enriched Tregs. The kit of the inventioncan also include reagents and instructions for assaying the expressionof various marker genes that can be used to characterize the Tregs. Forexample, these can include reagents and instructions for detecting themRNA or protein levels of one or more of FOXP3, CTLA4, TNFR2, CD62L,Fas, HLA-DR, CD45RO, CD127, CCR5, CCR6, CCR7, CXCR3, IFN-gamma, IL10,and ICOS.

The invention features a kit for the production of a compositionenriched in lymphocytes and depleted of Tregs. The kit can include aTNFR2 antagonist (e.g., a TNFR2 antagonist antibody) or an NF-κBinhibitor (e.g., one or more of the NF-κB inhibitors described above),reagents and/or devices for isolating a human blood or bone marrowsample, reagents and/or devices for isolating blood or bone marrow cells(e.g., T lymphocytes, such as CD4+ cells, CD25+ cells, or 004+0025+cells) from the sample, and reagents for culturing the blood cells(e.g., anti-CD3 antibody, anti-CD28 antibody, interleukin-2, and/orrapamycin). Additionally the kit of the invention can also includeinstructions for performing the method of the invention, e.g.,instructions for isolating a blood or bone marrow sample and blood orbone marrow cells therefrom, instructions for contacting blood cellswith a TNFR2 antagonist, and/or instructions for culturing, harvestingand/or storing the enriched lymphocytes. The kit of the invention canalso include reagents and instructions for assaying the expression ofvarious marker genes that can be used to characterize the lymphocytes.For example, these can include reagents and instructions for detectingthe mRNA or protein levels of one or more of FOXP3, TRAF2, TRAF3, andclAP.

The invention also features kits that include a composition containingan anti-TNFR2 antibody (e.g., an anti-TNFR2 antagonist antibody), apharmaceutically-acceptable carrier or excipient, and, optionally, otheragents as described herein; the composition contains an effective amountof the anti-TNFR2 antibody for treating a proliferative disease or aninfectious disease. The kits may include instructions explaining how apractitioner (e.g., a physician, nurse, or patient) may administer thecomposition contained therein. Furthermore, the kits may also includeadditional components, such as one or more additional componentsdescribed above, instructions or administration schedules for a patientsuffering from a proliferative disease or an infectious disease, and,optionally, a device(s) for administering the composition (e.g., asyringe).

The following examples are meant to illustrate the invention. They arenot meant to limit the invention in any way.

EXAMPLES Materials and Methods

Human Subjects and TNF-α Induction with BCG Vaccine

Two BCG vaccinations were used to induce TNF-a. Administration of BCGwas approved by the Human Studies Committee at Massachusetts GeneralHospital and by the FDA (NCT00607230).

For the double-blinded placebo-controlled trial, one subject wasinjected with BCG at a dose of 1.6-3.2×10⁶ cfu and the placebo subjectwas injected with saline. The BCG or saline injection was administeredintradermally on two occasions four-weeks apart. All blood samples wereblinded and simultaneously sent to the laboratory for monitoring TNF-αand Treg levels.

Reagents and Flow Cytometry

Recombinant human TNF-α was purchased from Leinco Technologies (St.Louis, Mo.), and recombinant human IL-2 was purchased from Sigma-Aldrich(St. Louis, Mo.). Monoclonal antibodies against TNFR1 and TNFR2 used forscreening purposes were from internal sources and external vendors(Table 1). External vendors included R&D Systems, Inc.,Hycult-Biotechnology, BD-Pharmingen, Accurate, Abcam and Sigma. Allother antibodies were purchased from BD-Biosciences. Intracellularstaining of FOXP3 and CD152 were performed using either FOXP3 Fix/PermBuffer set (Biolegend) or Human FOXP3 Buffer set (BD Biosciences). Theavidities of MAB726 and M1 antibodies for TNFR2 were determined usingsurface plasmon resonance on Pioneer SensiQ (SensiQ Technologies,Oklahoma City, Okla.).

TABLE 1 Clone MR2-1 80M2 MAB726 M1 MAB2261 Isotype Mouse Mouse Mouse RatIgG2b Mouse IgG1 IgG1 IgG1 IgG2A Properties Agonist Neutral AntagonistAntagonist Agonist Vendor Cell Cell R&D BD R&D Sciences SciencesBiosciencesCD4+ Cell isolation, induction of FOXP3, and expansion of CD4⁺CD25⁺cells

CD4+ T cells were isolated using Dynal CD4 Positive Isolation Kit(Invitrogen). Extraction of CD25 positive cells was subsequentlyperformed after CD4+ isolation using Dynabeads CD25 and DETACHaBEADCD4/CD8 (Invitrogen). After isolation, 2×10⁴ cells were cultured in96-round-bottom well plate. Dynabeads for human Treg Expander(Invitrogen) (Dynabeads coupled with anti-CD3 and anti-CD28 monoclonalantibodies) was added at a beads-to-cell ratio of 2:1. In selectedwells, TNF-α (20ng/ml), TNFR2 mAbs (2.5 μg/ml), rapamycin (1 μM, EMDBiosciences, San Diego, Calif.) were added. After two days, IL-2 (200U/ml) was added to the culture. Half of the media was changed every 2 to3 days containing rapamycin (until day 7) and 100 U/ml of IL-2. On day9, additional TNF-α or TNFR2 mAbs were supplied into the media. On day16, cells were harvested, Dynabeads Human Treg Expander was removed,washed and rested. On the following day, cells were analyzed.

Intracellular Staining

Expanded CD4+CD25⁺ cells were stimulated with phorbol myristate acetate(PMA) (2 ng/ml) and ionomycin (500 ng/ml) (Sigma) for 24 hours. Monensin(GolgiStop, BD Biosciences) was added for the last 4 hours ofincubation. Cells were fixed and permeabilized using Human FOXP3 BufferSet, followed by staining with fluorochrome-conjugated IFNγ and IL-10mAbs.

mRNA Isolation

Isolated CD4+ cells were incubated in presence of IL-2 (50 U/ml) with orwithout TNFR2 mAbs (2.5 μg/ml). After 3 hours, cells were collected andtotal RNA was isolated using RNAqueous-4PCR kit (Ambion, Austin, Tex.).The extracted RNA was reverse transcribed using High Capacity cDNAReverse Transcription Kit (Applied-Biosystems, Foster City, Calif.).

Cell Proliferation and Suppression Assays

For CD4 proliferation experiments, CD4+ cells were stained with 1 μMcarboxyfluorescein diacetate succinimidyl ester (CFSE). Cells wereplated at the density of 2×10⁵ cells/well in 96-well plate withanti-CD3mAbs . Four days later, cells were collected and analyzed.

For Treg suppression assay, autologous PBMCs were used as responders.PBMC were collected using Ficoll-Paque, cryopreserved at −80° C., andthawed the day before mixing with Tregs and rested overnight in RPMI1640 and 10 U/ml IL-2. The next day, responder cells were stained withCFSE (1 μM). Responder cells (5×10⁴ cells) and expanded Tregs were mixedat various ratios, and stimulated with anti-CD3mAb and IL-2. After 4days, cells were collected and analyzed.

Statistical Analysis

All data analyses were performed by the paired Student t test usingGraphPad Prism-5 software (GraphPad Software, La Jolla, Calif.). Iconsidered two-sided p value 0.05 as significant.

Example 1 Clinical Trial Induction of Human Tregs

Unexpanded, naturally occurring human Tregs are heterogeneous and rarein blood. Homogeneous populations of Tregs are difficult to expand invitro even with multiple ligand mixtures. With the goal of expandingsufficient numbers of homogeneous populations of human Treg cells TNF-α,I first sought to confirm or refute an increase in Treg concentrationsby induction with native TNF-α. Because an FDA-approved version of TNF-αdoes not exist and manufacturing of a stable form is difficult, Iadministered a well-known, strong inducer of TNF-α, Bacillus CalmetteGuerin (BCG), a generic vaccine already on the market for decades fortuberculosis and bladder cancer. This method of inducing endogenousTNF-α eliminated the manufacturing problems of TNF-α that formsunnatural monomers, dimers and trimers with differential cellulareffects.

The small, double-blinded placebo-controlled clinical trial enrolled twosubjects. One human subject received BCG injections (1.6-3.2×10⁶cfu/injection) and the placebo received saline, twice, 4-weeks apart.Both were monitored weekly for 20-weeks to study the pharmacokinetics ofTNF-α and Treg induction. After each injection TNF-α induced Tregs in abi-modal fashion with slightly delayed kinetics (FIG. 1A, left panels).After 20 weeks of observation, saline injections induced neither TNF-αnor Tregs. The total CD4+ cell counts did not change in BCG and placebopatients other than in the percentages of CD4+CD25^(hi)FOXP3+ depictedin FIG. 1A. This in vivo evidence confirms endogenous TNF-α in vivoincreases the numbers of Tregs but since TNF-α binds to both the TNFR1and TNFR2 receptors, it does not clarify which receptor was more centralfor the Tregs effect.

Example 2 Functional Effects of TNFR Monoclonal Antibodies and SignalingPathways

I cultured freshly isolated human CD4+ cells from 14 human subjects onlywith TNF-α for 16 hours (FIG. 1 B). While finding no induction of Tregs,assessed by inducible FOXP3, I observed a significant increase in Tregsafter adding IL-2. Co-incubation of TNF-α and IL-2 produced asignificant increase in Tregs over IL-2 alone (FIG. 1 B). IL-2 isimportant for Tregs induction and maintenance in mice. Findings wereconfirmed by flow cytometry, in which co-incubation increased thepercentage of CD4+CD25^(hi) FOXP3 cells in human cultured cells fromblood (FIG. 1 C). Thus, the data in FIGS. 1B and 1C confirm that TNF-αcan induce a homogeneous population of Tregs in vitro.

In freshly isolated CD4+ cells, I examined expression levels of eachTNFR in relation to CD25+ expression. TNFR1 expression on CD4+ cells,regardless of CD25+ expression levels, was unchanged using flowcytometry (FIG. 2A, middle panel), whereas TNFR2 preferentiallyexpressed CD4+CD25^(hi) Tregs by nearly a factor of 10 (FIG. 2A, rightpanel). This confirms earlier studies that TNFR2 is more denselyexpressed on Tregs.

Screening several TNFR1 and TNFR2 monoclonal antibodies (mAbs) onisolated CD4+ cells sampled from fresh human blood enabled selectivestudy of each TNF receptor, unlike studying TNF-α, which acts throughboth receptors and can have manufacturing problems from the use of E.coli and yeast systems. Although most of the screened TNFR1 or TNFR2mAbs failed to induce or suppress FOXP3+ Tregs after stimulation bypresence of IL-2 for 16 hours (FIGS. 7 and 8), I found two types ofTNFR2 mAbs with significant, and opposing, effects on FOXP3 induction(FIG. 2b ). Studying freshly cultured cells from 10 subjects, one TNFR2antibody significantly induced FOXP3 expression in CD4+ human T cells(which I designated the “TNFR2 agonist”), whereas the other TNFR2monoclonal suppressed intracellular FOXP3 expression (which I designatedthe “TNFR2 antagonist”) (FIG. 2B). Thus, I have identified M1 and MAB726as TNFR2 antagonists.

Having identified two functionally-opposing types of TNFR2 mAbs, Imeasured their effects on isolated CD4+ T cells by examining downstreammRNA expression in signaling proteins specific to TNFR2 activation.After 24 h stimulation by the TNFR2 agonist or antagonist, relative mRNAexpression was significantly different. The TNFR2 agonist stimulatedexpression of TNF, TRAF2, TRAF3, clAP2 and FOXP3. In contrast, the TNFR2antagonist stimulated expression of clAP1, but not TRAF2, TRAF3 or FOXP3(FIG. 2C).

The effects of the TNFR2 agonist and antagonist were studied on purifiedhuman CD4+ T cells co-cultured with anti-CD3 or IL-2. When CD4+proliferation was studied with anti-CD3 combined with the TNFR2 agonist,the highest degree of proliferation was shown by the agonist. Incontrast, the TNFR2 antagonist (e.g., M1 or MAB726) suppressed CD4+proliferation even relative to the control, anti-CD3 alone (FIG. 2D,left-most panel). The same findings were observed after 4 days bydirectly measuring CD4+ proliferation by flow cytometry and measuringCD4+ proliferation by CFSE dilution (FIG. 2D, three right-most panels).Thus, the opposing effects of TNFR2 agonist treatment versus TNFR2antagonist treatment on Tregs have been demonstrated, as shown in FIGS.2B-2D.

Despite high expression of TNFR2 on Tregs, some TNFR2 expression is alsoobservable on CD4+ T cells that are not true Tregs because they onlyexpress intermediate levels of CD25, i.e., CD4⁺CD25^(mid) cells. Itherefore studied the impact of overnight incubation on CD25^(mid) cellsubpopulations of IL-2 alone, IL-2 and TNF-α, or IL-2 and TNFR2 agonist,or IL-2 and TNFR2 antagonist. I found a rise in the proportion ofCD25^(hi)FOXP3⁻ cells similar to effector cells with IL-2 and TNF-αstimulation alone, or IL-2 and TNFR2 agonist alone (FIG. 8). However, Iobserved suppression with IL-2 and TNFR2 antagonist relative to theother three groups. Therefore, the TNFR2 agonist and antagonist, studiedby the same assay, showed opposing trends on the same CD25⁺FOXP3− cellpopulation. Thus, the addition of a TNFR2 antagonist (e.g., M1 orMAB726) inhibited expression of Foxp3 on CD4+CD25+ T cells.

I separated fresh human blood to obtain pure CD4+ and CD25^(hi)co-expressing Tregs (FIG. 3). I purified and expanded these Tregs invitro using standard protocols of anti-CD3 anti-CD28 plus IL-2 for 16days (FIG. 3A), then rested them overnight before counting. I addedrapamycin (until day 7) because it selectively expands the highestnumber of Tregs with greatest capacity for suppressing CD8+ cells. Thisprocess successfully produced CD4+CD25^(hi) Tregs (FIG. 3B). I assessedT_(reg) expansion by treatment group: no treatment, treatment withTNF-α, TNFR2 agonist, or TNFR2 antagonist. The TNFR2 agonistoutperformed every other group, expanding Tregs at least twofold higherthan no treatment or antagonist treatment. The latter suppressedexpansion because its effect was less than that of no treatment. Becauserapamycin is known to inhibit proliferation, I examined the effects oftreatment without rapamycin, yet found similarly opposing effectsbetween agonist versus antagonist treatment, albeit at smaller meanabsolute values (FIG. 9B). The yields of expanded cells tended to beless without rapamycin, but the agonist still expanded Tregs.

Example 3 TNFR2 Agonist Expansion and Homogeneity of Tregs

I next investigated whether in vitro Tregs, treated by TNFR2 agonist,possessed more homogeneous Treg cell surface markers than those treatedby the antagonist. Comparing phenotypes for 14 cell surface markers, alltreatment groups highly expressed Treg signature markers FOXP3 and CD25(FIG. 4A). The expression levels of FOXP3 were similar to levels beforetreatment. However, CD25+ expression was much higher after agonisttreatment, which can be considered an expansion effect rather than anantagonist effect (data not shown). Nearly 100% of expanded CD25^(hi)Tregs in each group were positive for CTLA4, TNFR2, CD62L, Fas, andnegative for CD127 (FIG. 4A). Tregs treated with TNFR2 antagonist alsomaintained expression for these markers. In contrast, several othersurface markers, such as HLA-DR, ICOS, CD45RO and chemokine receptors,were differentially expressed between the agonist vs. antagonisttreatment (FIGS. 4B and 4C and FIG. 10). Similar results were observedin Tregs expanded without rapamycin (FIG. 10). Tregs expanded by TNFR2agonist—relative to most other comparator groups, especially the TNFR2antagonist—yielded a surprisingly homogeneous population of cells withthis phenotype:CD4+CD25^(hi)FOXP3⁺CTLA4⁺TNFR2⁺CD127⁻CD62L⁺Fas⁺HLA-DR⁺CD45RO⁺CCR5⁻CCR6⁻CCR7⁻CXCR3⁻ICOS⁻.The mean fluorescence intensity (MFI), a direct measure of the averagedensity of the protein per cell, similarly revealed that for mostsurface markers, the TNFR2-agonist treated cells showed opposingexpression levels compared to TNFR2-antagonist treated cells (FIG. 12).Further investigation should define whether these expanded cellsmaintain phenotypic homogeneity over time but the evidence from thisconventional expansion protocol shows that they were more homogeneousthan other groups. Before treatment, Treg markers were moreheterogeneous (FIG. 11). Unexpanded, naturally occurring human Tregs areheterogeneous populations. In vitro studies of mixed Treg populations,which include CD45RO⁺FOXP3^(low) T cells, produce pro-inflammatorycytokines. This particular phenotype is found in up to 50% of FOXP3+ Tcells.

One of the most upregulated markers by TNFR2 agonist-treated Tregs wasHLA-DR, which is reported to have higher suppressive activity againstCD8 T+ cells, suggestive of an effector Treg. In contrast to HLA-DR, allfour chemokine receptors were strongly down-regulated. Although the lackof chemokine receptors might result in failure to migrate to the site ofinflammation, another homing receptor, CD62L, which was highly expressedin all treatment groups (FIG. 4), is crucial for entering the site ofpathogenic T cell presence in acute GVHD. The fact that agonist-treatedTregs were CD45RO+ and CCR7− and displayed significantly higherexpression levels of Fas, measured by MFI (FIG. 12), contributes to theview that they are activated effector Tregs.

Example 4 TNFR2 Agonist-Treated Tregs and CD8+ Suppression

One key function of Tregs, especially in autoimm unity, is to suppressthe function of autoreactive cytotoxic CD8+ T cells. I assessed thiscapacity by mixing Tregs from each treatment group with CFSE-stainedautologous PBMC, after having stimulated them with anti-CD3mAb and IL-2for 4 days. Autologous CD8+ T cells, the responder cells, were testedfor suppression by observing the ratios of responders to Tregs. Ratiosof dilution enable study of dose-dependence. All groups of Tregsdisplayed suppressive function on CD8+ T cells, but the degree varied bytreatment group (FIG. 5A, left panel). TNFR2 agonist-treated Tregs, forexample, showed the strongest suppressive capacity at 1:1 ratio (byleaving the fewest number of CD8+ cells) and then became progressivelyweaker at higher ratios. However, the antagonist-treated cells displayedweaker suppressive capacity that was essentially no different from thatof no treatment. With a suppression index of 2:1, the TNFR2agonist-treated group showed greater suppression than did the antagonistand no treatment groups (FIG. 5A, right panel). Similar results wereobserved with Tregs treated without rapamycin (FIG. 13A). The resultsare consistent with known phenotypes and expansion capacity offunctional Tregs.

Example 5 TNFR2 Agonist-Treated Tregs and Cytokine Production

I found that all treatment groups had relatively limited ability toproduce intracellular IFNγ and IL-10 after PMA and ionomycinstimulation. But TNFR2 agonist and the TNF only-treated Tregs producedthe lowest percentages of IFNγ+cells (FIG. 5B, lowest left panel). Theantagonist-treated Tregs showed significantly higher IFNγ productionthan the agonist (FIG. 5b , lower left panel). In similar experimentswithout rapamycin, the TNFR2 agonist-treated group not only producedlower IFNγ, but also lower IL-10 and TNF production relative to TNF orno treatment, respectively (FIG. 13B). Agonist-treated Tregs also showedthe fewest number of TH1 Transcription Factor (T-bet)+Tregs (FIG. 5C),which is consistent with lower IFNγ production (FIG. 5C). One of thereasons for these Tregs showing high suppression capacity over CD8+ Tcells may be due to Tregs lacking the ability to produce IFNγ.

Other Embodiments

The disclosures of U.S. Provisional Patent Application No. 61/762,136,filed on Feb. 7, 2013, and U.S. Provisional Patent Application No.61/763,217, filed on Feb. 11, 2013, are hereby incorporated by referencein their entirety.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated as being incorporated by reference in theirentirety.

Other embodiments are in the claims.

1-16. (canceled)
 17. A composition enriched in CD4+CD25hi Tregs, whereinsaid Tregs comprise at least 60% of the cells in said composition, andwherein said composition comprises at least 5×10⁶ Tregs. 18-20.(canceled)
 21. The composition of claim 17, wherein said Tregs comprisesubstantially 100% of the cells in said composition.
 22. The compositionof claim 21, wherein said Tregs are further characterized as positivefor the expression of one or more proteins selected from the groupconsisting of FOXP3, CTLA4, TNFR2, CD62L, Fas, HLA-DR, and CD45RO or lowor negative for the expression of one or more proteins selected from thegroup consisting of CD127, CCRS, CCD6, CXCR3, IFN-gamma, IL10, and ICOS.23-50. (canceled)
 51. An isolated, non-murine antibody orantigen-binding fragment thereof that selectively binds to an epitope ofTNFR2 within amino acids 130-149 of SEQ ID NO: 1, said antibody orantigen-binding fragment thereof having an antagonistic effect on TNFR2upon binding.
 52. (canceled)
 53. The antibody or antigen-bindingfragment thereof of claim 51, wherein said epitope comprises amino acids135-147 of SEQ ID NO:
 1. 54-57. (canceled)
 58. The antibody orantigen-binding fragment thereof of claim 51, wherein: i) said antibodyor antigen-binding fragment thereof is selected from the groupconsisting of a monoclonal antibody or antigen-binding fragment thereof,a polyclonal antibody or antigen-binding fragment thereof, an Fab, ahumanized antibody or antigen-binding fragment thereof, a bispecificantibody or antigen-binding fragment thereof, a monovalent antibody orantigen-binding fragment thereof, a chimeric antibody or antigen-bindingfragment thereof, a single-chain Fv molecule, a bispecific single chainFv ((scFv′)2) molecule, a domain antibody, a diabody, a triabody, anaffibody, a domain antibody, a SMIP, a nanobody, a Fv fragment, a Fabfragment, a F(ab′)2 molecule, and a tandem scFv (taFv) fragment; or ii)said antibody or antigen-binding fragment thereof exhibits anequilibrium dissociation constant for binding of said antibody orantigen-binding fragment thereof to TNFR2 of less than about 50 nM.59-61. (canceled)
 62. A method for producing a composition enriched inlymphocytes comprising contacting in vitro a population of human cellscomprising said lymphocytes obtained from a human blood or bone marrowsample from a patient with a tumor necrosis factor receptor 2 (TNFR2)antagonist or an NF-κB inhibitor that suppresses proliferation of Tregulatory cells (Tregs), thereby producing said composition enriched inlymphocytes, wherein said Tregs comprise less than 10% of the cells insaid composition. 63-64. (canceled)
 65. The method of claim 62, whereinsaid TNFR2 antagonist is selected from the group consisting of anantibody or antigen-binding fragment thereof, a peptide, a smallmolecule, and a protein or said NF-κB inhibitor is selected from thegroup consisting of a small molecule, a peptide, a protein, a virus, anda small non-coding RNA.
 66. (canceled)
 67. The method of claim 65,wherein said antibody or antigen-binding fragment thereof is amonoclonal anti-TNFR2 antagonist antibody or antigen-binding fragmentthereof or said NF-κB inhibitor is selected from the group consisting of2-(1,8-naphthyridin-2-yl)-Phenol, 5-Aminosalicylic acid, BAY 11-7082,BAY 11-7085, CAPE (Caffeic Acid Phenethylester), Diethylmaleate, Ethyl3,4-Dihydroxycinnamate, Helenalin, Gliotoxin, NF-κB Activation InhibitorII JSH-23, NFκB Activation Inhibitor III, Glucocorticoid ReceptorModulator, CpdA, PPM-18, Pyrrolidinedithiocarbamic acid ammonium salt,(R)-MG-132, Rocaglamide, Sodium Salicylate, QNZ, MG-132[Z-Leu-Leu-Leu-CHO], Astaxanthin, (E)-2-Fluoro-4′-methoxystilbene,CHS-828, disulfiram, olmesartan, triptolide, withaferin, celastrol,tanshinone IIA, Ro 106-9920, cardamonin, BAY 11-7821, PSI, HU 211,ML130, PR 39, honokiol, CDI 2858522, andrographolide, anddithiocarbamates.
 68. The method of claim 65, wherein said antibody orantigen-binding fragment thereof binds to an epitope of TNFR2 withinamino acids 130-149 of SEQ ID NO:
 1. 69. (canceled)
 70. The method ofclaim 68, wherein said epitope comprises amino acids 135-147 of SEQ IDNO:
 1. 71. The method of claim 68, wherein: i) said antibody orantigen-binding fragment thereof is selected from the group consistingof a monoclonal antibody or antigen-binding fragment thereof, apolyclonal antibody or antigen-binding fragment thereof, an Fab, ahumanized antibody or antigen-binding fragment thereof, a bispecificantibody or antigen-binding fragment thereof, a monovalent antibody orantigen-binding fragment thereof, a chimeric antibody or antigen-bindingfragment thereof, a single-chain Fv molecule, a bispecific single chainFv ((scFv′)2) molecule, a domain antibody, a diabody, a triabody, anaffibody, a domain antibody, a SMIP, a nanobody, a Fv fragment, a Fabfragment, a F(ab′)2 molecule, and a tandem scFv (taFv) fragment; or ii)said antibody or antigen-binding fragment thereof exhibits anequilibrium dissociation constant for binding of said antibody orantigen-binding fragment thereof to TNFR2 of less than about 50 nM.72-79. (canceled)
 80. A composition produced by the method of claim 62,wherein said composition is enriched in lymphocytes, and wherein Tregsin said composition comprise less than 10% of the cells in saidcomposition.
 81. (canceled)
 82. A method of treating a proliferativedisorder or an infectious disease in a patient comprising administeringto said patient an isolated antibody or antigen-binding fragment thereofthat selectively binds to TNFR2 as an antagonist.
 83. The method ofclaim 82, wherein said proliferative disorder is a cancer selected fromthe group consisting of Acute Lymphoblastic Leukemia, AcuteLymphoblastic Leukemia, Acute Myeloid Leukemia, AdrenocorticalCarcinoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Osteosarcoma/Malignant Fibrous Histiocytoma, Brain Stem Glioma, VisualPathway and Hypothalamic Glioma, Breast Cancer, BronchialAdenomas/Carcinoids, Chronic Lymphocytic Leukemia, Chronic MyelogenousLeukemia, Chronic Myeloproliferative Disorders, Clear Cell Sarcoma ofTendon Sheaths, Colon Cancer, Colorectal Cancer, Cutaneous T-CellLymphoma, Endometrial Cancer, Epithelial Cancer, Esophageal Cancer,Ewing's Family of Tumors, Extracranial Germ Cell Tumor, ExtragonadalGerm Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, IntraocularMelanoma, Retinoblastoma, Gallbladder Cancer, Gastric (Stomach) Cancer,Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular (Liver)Cancer, Hodgkin's Lymphoma, Hypopharyngeal Cancer, Kaposi's Sarcoma,Kidney Cancer, Laryngeal Cancer, Pituitary Cancer, Plasma CellNeoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Skin Cancer, SmallCell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, SquamousNeck Cancer, Testicular Cancer, Thyroid Cancer, Urethral Cancer, UterineSarcoma, and Vaginal Cancer, or wherein said proliferative disorder is asolid tumor of the brain, lung, breast, lymphoid, gastrointestinaltract, genitourinary tract, pharynx, prostate, or ovary. 84-85.(canceled)
 86. The method of claim 82, wherein said infectious diseaseis selected from the group consisting of a bacterial infection, a viralinfection, a fungal infection, and a parasitic infection. 87-90.(canceled)
 91. The method of claim 82, wherein said antibody orantigen-binding fragment thereof is a non-murine antibody orantigen-binding fragment thereof that selectively binds to an epitope ofTNFR2 within amino acids 130-149 of SEQ ID NO:
 1. 92. A method oftreating a proliferative disorder or an infectious disease in a patientcomprising administering to said patient the composition of claim 80.